Regulating alkaloids

ABSTRACT

MPO1 and MPO2 can be regulated for either decreasing or increasing alkaloid levels in plants, in particular in  Nicotiana  plants. In particular, suppressing or overexpressing one or more of MPO1 and MPO2 may be used to decrease or increase nicotine and nicotinic alkaloid levels in tobacco plants. Suppression or overexpression of one or more of MPO1 and MPO2 may be used in combination with modification of expression of other genes encoding enzymes on the nicotinic alkaloid biosynthetic pathway such as A622, NBB1, PMT, and QPT.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.60/866,352, filed Nov. 17, 2007.

FIELD OF THE INVENTION

The present invention relates to the field of molecular biology andregulation of alkaloid synthesis. More specifically, the inventionrelates to regulating alkaloid content in a plant, particularly but notexclusively nicotinic alkaloids in a tobacco plant.

BACKGROUND

Genes encoding nicotine biosynthesis enzymes are known. For example, thetobacco quinolate phosphoribosyl transferase (QPT) gene has been cloned;see U.S. Pat. No. 6,423,520 and Sinclair et al., Plant Mol. Biol. 44:603-17 (2000). QPT suppression provides significant nicotinic alkaloidreductions in transgenic tobacco plants. Xie et al., Recent Advances inTobacco Science 30: 17-37 (2004). Likewise, suppression of an endogenousputrescine methyl transferase (PMT) sequence has been shown to reducenicotine levels but increase anatabine levels by about 2-to-6-fold. Hibiet al., Plant Cell 6: 723-35 (1994); Chintapakom and Hamill, Plant Mol.Biol. 53:87-105 (2003); Steppuhn et al. PLoS Biol2:8:e217: 1074-1080(2004). Levels of nicotine and other nicotinic alkaloids are reduced intobacco by suppressing either the A622 or NBB1 nicotine biosynthesisgenes. See WO/2006/109197.

Despite this, a comprehensive understanding of how the nicotinebiosynthetic pathway functions is essential. Accordingly, furtherresearch efforts have been underway to elucidate the biochemistry andmolecular biology of this pathway, including the identification of allrelated genes. Additional insights into biosynthesis pathways of othernicotinic alkaloids and of other alkaloids found in non-Nicotiana plantswould be facilitated by a comprehensive understanding of the nicotinebiosynthesis pathway in tobacco.

Reducing total alkaloid content in tobacco would increase the value oftobacco as a biomass resource. Reduced-alkaloid tobacco is more amenablefor non-traditional purposes, such as biomass and derived products. Forexample, it is advantageous to use reduced-alkaloid tobacco forproducing ethanol and protein co-products. Additionally, alkaloid-freetobacco or fractions thereof may be used as a forage crop, animal feed,or a human nutritive source. See WO/2002/098208.

An additional use of reduced-nicotine tobacco is for smoking cessation.Nicotine-reduced or nicotine-free tobacco cigarettes have assistedsmokers in quitting smoking. Additionally, denicotinized cigarettesrelieve craving and other smoking withdrawal symptoms. See Rose,Psychopharmacology 184: 274-285 (2006) and Rose et al., Nicotine TobaccoRes. 8: 89-101 (2006).

It may be beneficial to overexpress a nicotine biosynthesis gene, asmeans for increasing nicotine biosynthesis and accumulation in tobacco.For example, because nicotinic alkaloids play an important role inprotecting plants against insects and herbivores, it is likely to beadvantageous to increase nicotinic alkaloid synthesis in a host plant.From an herbivory perspective, increased nicotine synthesis andaccumulation would provide an environmentally acceptable means formediating plant-pest interactions.

As nicotine is the physically and psychologically active component incigarette smoke, it may be advantageous to increase nicotine content intobacco by genetic engineering. Research studies demonstrate that whensupplementary nicotine is added to cigarette tobacco from an outsidesource, smokers inhale less of the more harmful components of smoke suchas tar and carbon monoxide. See Armitage et al., Psychopharmacology 96:447-53 (1988), Fagerström, Psychopharmacology 77: 164-67 (1982),Russell, Nicotine and Public Health 15: 265-84 (2000), and Woodman etal., European Journal of Respiratory Disease 70: 316-21 (1987).Likewise, a report by The Institute of Medicine of the U.S. on potentialreduced exposure products (PREPS) concluded that “retaining nicotine atpleasurable or addictive levels while reducing the more toxic componentsof tobacco is another general strategy for harm reduction.” See CLEARINGTHE SMOKE. ASSESSING THE SCIENCE BASE FOR TOBACCO HARM REDUCTION, 10M atpage 29 (2001), commonly referred to as the “IOM Report” by the tobaccoindustry.

The part of the nicotine biosynthesis pathway that produces theN-methylpyrrolinium cation also is part of the pathway for thebiosynthesis of other alkaloids, including medicinal tropane alkaloids.Hashirnoto and Yamada, Annu. Rev. Plant Physiol. Plant Mol. Biol. 45:257-285 (1994); Kutchan, T. M., “Molecular genetics of plant alkaloidbiosynthesis. In: Cordell, G. A. (ed.) 50 ALKALOIDS 257-316 (AcademicPress, 1998).

A plant also can be genetically engineered to regulate its alkaloidprofile, such as the ratio of a particular alkaloid to total alkaloidcontent. For example, if the goal is increasing the ratio of anatabineto total alkaloid content of a N. tabacum plant, PMT is suppressed.Chintapakorn and Hamill, supra.

As more alkaloid biosynthesis genes are discovered, including anunderstanding of their function and location in alkaloid biosynthesispathways, the more sophisticated genetic engineering of these pathwayscan become. Accordingly, there is a continuing need to identifyadditional genes whose expression can be regulated to not only decreaseor increase alkaloid(s) but to alter a plant's alkaloid profile, inparticular, nicotinic alkaloids in N. tabacum plants.

SUMMARY OF THE INVENTION

The present inventors have identified two genes, MPO1 and MPO2, whichcan be regulated independently or simultaneously to achieve a decreaseor increase of nicotine levels in Nicotiana plants, and to achieve adecrease or increase of tropane alkaloids in Erythroxylaceae,Solanaceae, and Convolvulaceae plant families.

In one aspect, the invention provides an isolated nucleic acid moleculecomprising a nucleotide sequence selected from the group consisting of:(a) a nucleotide sequence set forth in SEQ ID NO: 1; (b) a nucleotidesequence set forth in SEQ ID NO: 3; (c) a nucleotide sequence comprisingat least 15 consecutive nucleotides of the nucleotide sequence set forthin SEQ ID NO: 1 or 3; (d) a nucleotide sequence that encodes apolypeptide having the amino acid sequence set forth in SEQ ID NO: 2 or4; and (e) a nucleotide sequence that encodes a polypeptide having anamino acid sequence with at least 80% similarity to at least one of SEQID NO: 2 and 4 and that has MPO activity. In one embodiment, a nucleicacid construct comprising the nucleic acid molecule, wherein the nucleicacid is operatively linked in sense, antisense, or inverted repeatorientation to a heterologous promoter. In a further embodiment, a plantcell comprises the nucleic acid construct. In another embodiment, thereis provided a method of producing a reduced-alkaloid plant, comprisinggenetically engineering MPO suppression in the plant, wherein theengineering comprises introducing into a plant cell of the plant thenucleic acid construct. In a further embodiment, the plant is tobacco.In another embodiment, a tobacco plant comprises a chimeric nucleic acidconstruct, wherein the construct comprises the nucleic acid linked to aheterologous nucleic acid.

In another aspect, the invention provides a nucleic acid construct,comprising, in the 5′ to 3′ direction, a promoter operably linked to aheterologous nucleic acid encoding at least a portion of MPO1 or MPO2 insense, antisense, or inverted repeat orientation, and a terminator. Inan embodiment, a plant cell comprises the nucleic acid construct. Inanother embodiment, there is provided a method of producing areduced-alkaloid plant, comprising genetically engineering MPOsuppression in the plant, wherein the engineering comprises introducinginto a plant cell of the plant the nucleic acid construct. In a furtherembodiment, the plant is tobacco.

In another aspect, the invention provides a mutational vector comprisinga sequence of oligonucleotides targeting a region comprising 15consecutive nucleotides of SEQ ID NO: 1 or 3.

In further embodiment, a method is provided for producing areduced-alkaloid plant, comprising genetically engineering MPOsuppression in the plant, wherein the engineering comprises introducinginto a plant cell of the plant the aforementioned nucleic acidconstruct.

In another aspect, the invention provides a method for reducing analkaloid in a tobacco plant, comprising (a) genetically engineering MPOsuppression in the plant; and (b) suppressing at least one additionalnicotine biosynthesis enzyme selected from the group consisting ofaspartate oxidase, quinolinate synthase, quinolate phosphoribosyltransferase, ornithine decarboxylase, putrescine N-methyltransferase,A622, and NBB1.

In another aspect, the invention provides a tobacco plant havinggenetically engineered suppression of MPO and reduced content ofnicotine. In one embodiment, the there is provided progeny of thetobacco plant, wherein the progeny have MPO suppression. In anotherembodiment, the invention provides seeds from the tobacco plant. Inanother embodiment, there is provided a reduced-alkaloid tobacco productcomprising a portion of the tobacco plant. In a further embodiment, theproduct is a cigarette. In another further embodiment, there is provideda smoking cessation product comprising a portion of the tobacco plant.

In another aspect, the invention provides a reduced-alkaloid tobaccoproduct produced from genetically engineered tobacco having suppressedMPO, wherein the product has a reduced collective amount ofN′-nitrosonornicotine (NNN),4-methylnitrosoamino-1-(3-pyridyl)-1-butanone (NNK), N′-nitrosoanatabine(NAT) and N′-nitrosoanabasine (NAB) compared to a similar tobaccoproduct prepared from a non-genetically engineered control tobaccoplant. In one embodiment, the product is a cigarette.

In another aspect, the invention provides an MPO1 polypeptide having theamino acid sequence of SEQ ID NO: 2

In another aspect, the invention provides an MPO2 polypeptide having theamino acid sequence of SEQ ID NO: 4

In another aspect, there is provided an isolated MPO enzyme encoded by anucleic acid sequence selected from the group consisting of: (a) anucleotide sequence set forth in SEQ ID NO: 1; (b) a nucleotide sequenceset forth in SEQ ID NO: 3; (c) nucleic acid sequences which hybridize toat least one of SEQ ID NO: 1 and SEQ ID NO: 3 under moderate stringencyor high stringency conditions and encode an MPO enzyme; and (d) nucleicacid sequences which differ from the nucleic acid sequence of (a), (b),or (c) above due to the degeneracy of the genetic code and encode an MPOenzyme.

In another aspect, the invention provides a method of producing an MPOenzyme, comprising (a) introducing an isolated nucleic molecule encodingat least one of MPO1 and MPO2 into a cell; and (b) growing the cellunder conditions such that MPO enzyme is produced. In one embodiment,the cell is selected from the group consisting of bacteria, yeast,filamentous fungi, algae, green plants, and mammalian cells.

In another aspect, the invention provides a method for increasingalkaloids in a plant, comprising overexpressing MPO relative to acontrol plant. In one embodiment, the method further comprisesoverexpressing PMT. In another embodiment, an increased alkaloid plantis produced by the these methods. In another embodiment, an increasednicotine plant is produced by the these methods. In a furtherembodiment, an increased nicotine product is produced from the increasednicotine plant. In a still further embodiment, the product is selectedfrom the group consisting of a cigarette, a pharmaceutical, and anutraceutical.

In another aspect, the invention provides a method for increasingnicotine in a Nicotiana plant, comprising overexpressing at least one ofMPO1 and MPO2 relative to a control plant. In one embodiment, the methodfurther comprises overexpressing at least one of QPT, PMT, A622, andNBB1. In another embodiment, an increased alkaloid plant is produced bythe these methods. In another embodiment, an increased nicotine plant isproduced by the these methods. In a further embodiment, an increasednicotine product is produced from the increased nicotine plant. In astill further embodiment, the product is selected from the groupconsisting of a cigarette, a pharmaceutical, and a nutraceutical.

In another aspect, the invention provides a method for increasingnicotine and yield in a Nicotiana plant, comprising: (a) crossing anincreased nicotine Nicotiana plant of claim 27 with a high yieldingNicotiana plant; and (b) selecting a progeny plant with increasednicotine and high yield. In an embodiment, an increased nicotine andyield plant is produced by the method.

In another aspect, the invention provides a method for increasingnicotine and yield in a Nicotiana plant, comprising (a) introducing intoa Nicotiana plant a construct comprising, in the 5′ to 3′ direction, apromoter operably linked to a heterologous nucleic acid encoding anenzyme that increases yield; (b) regenerating transgenic Nicotianaplants from the plant; and (c) selecting a transgenic Nicotiana planthaving increased nicotine content and increased yield relative to acontrol plant. In an embodiment, an increased nicotine and yield plantis produced by the method.

In another aspect, the invention provides a method for increasing theratio of nicotine to total alkaloids in a tobacco plant, comprising: (a)overexpressing at last one of MPO1 and MPO2; (b) overexpressing PMT; and(c) suppressing QPT. In one embodiment, there is provided a plantproduced by the method. In a further embodiment, a product withincreased nicotine to total alkaloids is produced from the plant.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A: Depicts a model of the nicotine biosynthesis pathway. Theabbreviations are: MPO1=methylputrescine oxidase-1,MPO2=methylputrescine oxidase-2, PMT=putrescine N-methyltransferase,QPT=quinolinate phosphoribosyl transferase. A662 and NBB1 are theproducts of genes recently identified as nicotine biosynthesis genes.See WO/2006/109197.

FIG. 1B: Depicts a model of the biosynthetic pathway of Scopolamine andcocaine.

FIG. 2: Depicts an RNA gel blot analysis of expression of MPO and PMT intobacco lines with different alleles at the NIC loci and in response tomethyljasmonate treatment.

FIG. 3: Depicts an RNA gel blot analysis of expression of MPO and PMT indifferent tissues of tobacco plants. rRNA indicates RNA blots probedwith a ribosomal RNA control sequence.

FIG. 4: Relative expression levels of MPO1 and MPO2 in tobacco hairyroots.

FIG. 5: Depicts a phylogenetic tree constructed using full length cDNAsequences of tobacco N-methylputrescine oxidases, plant diamineoxidases, and Arabidopsis homologues.

FIG. 6: T-DNA regions of pBI-MPO-Ri, pBI-MPO, pTobRD2-MPO, andpTPoxTMoxTQRi.

FIG. 7A: MPO and PMT mRNA levels in tobacco hairy roots lines.

FIG. 7B: MPO1, MPO2, PMT and QPT mRNA levels in MPO down-regulatedtobacco hairy roots.

FIG. 8: MPO activity in MPO down-regulated tobacco hairy root lines.

FIG. 9: Alkaloid levels in MPO down-regulated tobacco hairy root lines.

FIG. 10A: MPO mRNA in BY-2 cells transformed with pBI-MPO.

FIG. 10B: MPO enzyme activity in BY-2 cells transformed with pBI-MPO.

FIG. 11: Alkaloid levels in BY-2 cells transformed with pBI-MPO.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have cloned two N-methylputrescine oxidase (MPO)genes, N-methylputrescine oxidase-1 (MPO1) and N-methylputrescineoxidase-2 (MPO2). The nucleic acid sequence of MPO1, SEQ ID NO: 1, hasbeen determined and encodes the polypeptide sequence set forth in SEQ IDNO: 2. The nucleic acid sequence of MPO2, SEQ ID NO: 3, has beendetermined and encodes the polypeptide sequence set forth in SEQ ID NO:4. The nucleotide sequences of the MPO1 ORF and the MPO2 ORF are setforth in SEQ ID NO: 5 and SEQ ID NO: 6, respectively. All polynucleotideand polypeptide sequences of SEQ ID NO: 1 through SEQ ID NO: 6,including all respective variants thereof, are an object of the presentinvention.

MPO is a specific type of diamine oxidase (EC 1.4.3.6) that catalyzesthe oxidative deamination of N-methylputrescine to 4-methylaminobutanal,which spontaneously cyclizes to N-methylpyrrolinium cation. Hashimoto,et al., Plant Phsyiol. 93: 216-221 (1990). Tobacco MPO enzymes have beenpartially purified from the roots of N. tabacum and N. rustica, and wereshown to oxidize N-methylputrescine more efficiently than putrescine andcadaverine Mizusaki et al., Phytochemistry 11: 2757-2762 (1972); Fethand Wagner, Phytochemistry 24: 1653-1655 (1985); Davies et al.,Phytochemistry 28: 1573-1578 (1989); Walton and McLauchlan,Phytochemistry 29: 1455-1457 (1990); Haslam and Young, Phytochemistry31: 4075-4079 (1992); McLauchlan et al., Planta 191: 440-445 (1993).

Inhibitor studies suggest that MPO contains copper and pyrroloquinolinequinone, as found for typical diamine oxidases. Davies, et al.,Phytochemistry 28: 1573-1578 (1989). Roots of tobacco nic mutants, whichare defective in regulation of nicotine biosynthesis, contained somewhatlower MPO activity than wild-type tobacco roots, indicating that the NICregulatory loci might regulate MPO gene expression, although the NICregulation of MPO activity was not as clear as the tight regulationfound for putrescine N-methyltransferase (PMT) activity. Saunders andBush, Plant Physiol. 64: 236-240 (1979). Immunological studies using aputative MPO antiserum suggest that MPO is associated withS-adenosylhomocysteine hydrolase as part of a larger multi-enzymecomplex Heim and Jelesko, Plant Mol. Biol. 56: 299-308 (2004). However,direct biochemical evidence was missing. Until now, MPO genes have notbeen molecularly cloned from tobacco and other solanaceous plants thatsynthesize N-methylputrescine-derived alkaloids.

Four lines of evidence indicate that the inventive MPO genes, MPO1 andMPO2, are involved in the oxidation of methylputrescine and nicotinebiosynthesis in tobacco. First, the MPO1 and MPO2 genes are regulated bythe nicotine regulatory NIC loci, in the same way as other genesencoding nicotine biosynthetic enzymes, such as PMT, QPT, A622, andNBB1. Second, the MPO1 and MPO2 genes are expressed exclusively intobacco roots, as are PMT, QPT, A622, and NBB1. Third, like PMT, QPT,A622, and NBB1, MPO1 and MPO2 expression is up-regulated by treatmentwith methyljasmonate. Fourth, recombinant MPO1 oxidizesN-methylputrescine more efficiently than putrescine and cadaverine. Theenzymatic property is the same for MPO enzymes partially purified fromroots of N. tabacum and N. rustica. Thus, the two novel genes, MPO1 andMPO2, identified and cloned by the inventors, have a critical roleduring nicotine biosynthesis in Nicotiana.

Plants of the Solanaceae, Erythroxylaceae, and Convolvulaceae synthesizeN-methylputrescine-derived alkaloids, such as nicotine, hyoscyamine,scopolamine, atropine and cocaine. Accordingly, the present inventionencompasses both methodology and constructs for regulating alkaloids ina plant, by increasing or decreasing at least one of MPO1 and MPO2. Thatis, levels of nicotine and some other nicotinic alkaloids in tobacco canbe decreased or increased by regulating at least one of MPO1 and MPO2.Pursuant to this aspect of the invention, a plant, or any part thereof,is transformed with a nucleotide sequence, expression of whichsuppresses or up-regulates at least one of MPO1 and MPO2 and reduces orincreases alkaloid content, respectively.

DEFINITIONS

All technical terms employed in this specification are commonly used inbiochemistry, molecular biology and agriculture; hence, they areunderstood by those skilled in the field to which this inventionbelongs. Those technical terms can be found, for example in: MOLECULARCLONING: A LABORATORY MANUAL, 3rd ed., vol. 1-3, ed. Sambrook andRussel, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,2001; CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, ed., Ausubel et al.,Greene Publishing Associates and Wiley-Interscience, New York, 1988(with periodic updates); SHORT PROTOCOLS IN MOLECULAR BIOLOGY: ACOMPENDIUM OF METHODS FROM CURRENT PROTOCOLS IN MOLECULAR BIOLOGY,5^(th) ed., vol. 1-2, ed. Ausubel et al., John Wiley & Sons, Inc., 2002;GENOME ANALYSIS: A LABORATORY MANUAL, vol. 1-2, ed. Green et al., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1997; TOBACCO:PRODUCTION, CHEMISTRY AND TECHNOLOGY, D. L. Davis and M. T. Nielson(eds.); Wiley, 1999.

Methodology involving plant biology techniques are described here andalso are described in detail in treatises such as METHODS IN PLANTMOLECULAR BIOLOGY: A LABORATORY COURSE MANUAL, ed. Maliga et al., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1995. Varioustechniques using PCR are described, for example, in Innis et al., PCRPROTOCOLS: A GUIDE TO METHODS AND APPLICATIONS, Academic Press, SanDiego, 1990 and in Dieffenbach and Dveksler, PCR PRIMER: A LABORATORYMANUAL, 2^(nd) ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 2003. PCR-primer pairs can be derived from known sequencesby known techniques such as using computer programs intended for thatpurpose, e.g., Primer, Version 0.5, 1991, Whitehead Institute forBiomedical Research, Cambridge, Mass. Methods for chemical synthesis ofnucleic acids are discussed, for example, in Beaucage & Caruthers,Tetra. Letts. 22: 1859-62 (1981), and Matteucci & Caruthers, J. Am.Chem. Soc. 103: 3185 (1981).

Restriction enzyme digestions, phosphorylations, ligations, andtransformations were done as described in Sambrook et al., MOLECULARCLONING: A LABORATORY MANUAL, 2nd ed. (1989), Cold Spring HarborLaboratory Press. All reagents and materials used for the growth andmaintenance of bacterial cells were obtained from Aldrich Chemicals(Milwaukee, Wis.), DIFCO Laboratories (Detroit, Mich.), Invitrogen(Gaithersburg, Md.), or Sigma Chemical Company (St. Louis, Mo.) unlessotherwise specified.

The terms “encoding” and “coding” refer to the process by which a gene,through the mechanisms of transcription and translation, providesinformation to a cell from which a series of amino acids can beassembled into a specific amino acid sequence to produce an activeenzyme. Because of the degeneracy of the genetic code, certain basechanges in DNA sequence do not change the amino acid sequence of aprotein. It is therefore understood that modifications in the DNAsequences encoding MPO1 and MPO2, respectively, which do notsubstantially affect the functional properties of either enzyme arecontemplated.

Gene refers to a polynucleotide sequence that comprises control andcoding sequences necessary for the production of a polypeptide orprecursor. The polypeptide can be encoded by a full-length codingsequence or by any portion of the coding sequence. A gene may constitutean uninterrupted coding sequence or it may include one or more introns,bound by the appropriate splice junctions. Moreover, a gene may containone or more modifications in either the coding or the untranslatedregions that could affect the biological activity or the chemicalstructure of the expression product, the rate of expression, or themanner of expression control. Such modifications include, but are notlimited to, mutations, insertions, deletions, and substitutions of oneor more nucleotides. In this regard, such modified genes may be referredto as “variants” of the “native” gene.

As is conventional in the art, nucleotide sequences may be denoted byitalicized font (e.g., PMT), whereas polypeptide sequences are notitalicized (e.g., PMT).

NIC1 and NIC2 loci are two independent genetic loci in N. tabacum,formerly designated as A and B. Mutations nic1 and nic2 reduceexpression levels of nicotine biosynthesis enzymes and nicotine content,generally the nicotine content of wild type>homozygous nic2>homozygousnic1>homoyzgous nic1 and homozygous nic2 plants. Legg & Collins, Can. JCyto. 13:287 (1971); Hibi et al., Plant Cell 6: 723-735 (1994); Reed &Jelesko, Plant Science 167: 1123 (2004).

As used herein, “expression” denotes the production of the proteinproduct encoded by a nucleotide sequence. “MPO1 expression” refers tobiosynthesis of a gene product encoded by SEQ ID NO: 1. “MPO2expression” refers to biosynthesis of a gene product encoded by SEQ IDNO: 3. “MPO expression” refers to biosynthesis of a gene product encodedby SEQ ID NO: 1 and biosynthesis of a gene product encoded by SEQ ID NO:3.

The terms “suppression” or “reduction” or “down-regulation” are usedsynonymously to indicate that expression of a particular gene sequence,or variant thereof, in a cell or plant, including all progeny plantsderived thereof, has been reduced, relative to a control cell or plant.

MPO1 suppression denotes a reduction of MPO1 expression in an organismsuch as a cell or plant, relative to a control cell or plant. MPO1suppression includes the biosynthesis of a gene product encoded by SEQID NO: 1, any related ORF sequence, and all MPO1 polynucleotidevariants.

MPO2 suppression denotes a reduction of MPO2 expression in an organismsuch as a cell or plant, relative to a control cell or plant. MPO2suppression includes the biosynthesis of a gene product encoded by SEQID NO: 3, any related ORF sequence, and all MPO2 polynucleotidevariants.

MPO suppression refers to a reduction of both MPO1 and MPO2 expressionin the same organism such as a cell or plant, relative to a control cellor plant.

MPO1 and MPO2 belong to the sub-group of the larger enzyme family ofcoppercontaining diamine oxidases. Typically, these “regular” diamineoxidases prefer symmetric diamines over N-methylated diamines forsubstrates. Although the DNA sequences of regular tobacco diamineoxidases are not known, it is likely that several nucleotide segmentsare common to MPO1 or MPO2 and regular tobacco diamine oxidases. If afull-length MPO-encoding polynucleotide, MPO ORF, or an MPO nucleotidefragment of at least 15 nucleotides, or variants of any the foregoing,is utilized for the suppression of one or both of the MPO genes,suppression of regular diamine oxidase genes may also occur.

It is therefore an object of the present invention, that if MPOsuppression is desired without the co-suppression of the regular diamineoxidases, segments of the 3′- or 5′-untranslated regions of MPO1 and/orMPO2 genes are utilized for these specific sequences in tandem toprecisely target the MPO gene(s). Co-suppression of related-familymember genes and specific suppression of target genes by gene-specificregions have been reported in RNAi suppression of rice Rac gene familymembers. See Miki D, Itoh R, Shimamoto K, Plant Physiol. 138: 1903-1913,2005, which is incorporated herein by reference.

“Overexpression” or “up-regulation” or “increased expression” are usedsynonymously to indicate that expression of a particular gene sequence,or variant thereof, in a cell or plant, including all progeny plantsderived thereof, has been increased, relative to a control cell orplant.

“MPO1 overexpression” denotes an increasing of MPO1 expression in anorganism such as a cell or plant, relative to a control cell or plant.MPO1 overexpression includes the biosynthesis of a gene product encodedby SEQ ID NO: 1, any related ORF sequence, and all MPO1 polynucleotidevariants.

“MPO2 overexpression” denotes an increasing of MPO2 expression in anorganism such as a cell or plant, relative to a control cell or plant.MPO2 overexpression includes the biosynthesis of a gene product encodedby SEQ ID NO: 3, any related ORF sequence, and all MPO2 polynucleotidevariants.

“MPO overexpression” refers to an increase of both MPO1 and MPO2expression in the same organism such as a cell or plant, relative to acontrol cell or plant.

MPO1, MPO2, and MPO suppression and overexpression have the ability toregulate (decrease or increase) nicotine biosynthesis in N. tabacum, themost preferred plant. MPO suppression and overexpression also have theability to regulate (decrease or increase) nicotine biosynthesis inother nicotine-producing plants. Preferred nicotine-producing plantsinclude Nicotiana, Duboisia, Anthocercis and Salpiglessis genera in theSolanaceae or the Eclipta and Zinnia genera in the Compositae.

An “alkaloid” is a nitrogen-containing basic compound found in plantsand produced by secondary metabolism. A “nicotinic alkaloid” is nicotineor an alkaloid that is structurally related to nicotine and that issynthesized from a compound produced in the nicotine biosynthesispathway. As used herein and in the case of tobacco, “nicotinic alkaloidcontent,” “total alkaloid content” and “total alkaloids” are synonymousand mean the total levels of alkaloids found in a tobacco plant, forexample, in terms of pg/g dry weight (DW). For non-tobacco plants “totalalkaloid content” and “total alkaloids” are synonymous and mean thetotal levels of alkaloids found in a plant, for example, in terms ofpg/g dry weight (DW).

“Alkaloid biosynthesis genes,” are genes that encode alkaloidbiosynthesis enzymes and many are known. Additionally, about 12,000chemical structures are currently known as alkaloids. Exemplary alkaloidbiosynthesis enzymes include Tropinone reductase-I, Tropinonereductase-II and Hyoscyamine 6b-hydroxylase, which are involved intropane alkaloid biosynthesis. Hashimoto and Yamada, Current Opinion inBiotechnology, 14: 163-168 (2003), which is incorporated herein byreference.

Illustrative nicotinic alkaloids include but are not limited tonicotine, nornicotine, anatabine, anabasine, anatalline,N-methylanatabine, N-methylanabasine, myosmine, anabaseine,N′-formylnornicotine, nicotyrine, and cotinine. Other very minornicotinic alkaloids in tobacco leaf are reported, for example, in Hecht,S. S. et al., Accounts of Chemical Research 12: 92-98 (1979); Tso, T.C., Production, Physiology and Biochemistry of Tobacco Plant. IdealsInc., Beltsville, Md. (1990). The chemical structures of severalalkaloids are presented, for example, in Felpin et al., J. Org. Chem.66: 6305-6312 (2001).

“Anatabine” is formed in tobacco from two molecules of a metabolite ofnicotinic acid. Leete and Slattery, J. Am. Chem. Soc. 98: 6326-6330(1976). Since this proposed biosynthetic pathway does not involve adiamine oxidation step, diamine oxidase or MPO do not seem to beinvolved in the formation of anatabine. Previous studies havedemonstrated that PMT suppression reduces nicotine content but increasesputrescine and anatabine levels. Chintapakorn & Harnill, Plant Mol.Biol. 53: 87-105 (2003); Sato et al., Proc. Natl. Acad. Sci. USA 98,367-372. (2001); Steppuhn, A., et al., PLoS Biol 2(8): e217: 1074-1080(2004). Accordingly, the MPO genes of the present invention likely donot contribute to the formation of anatabine. Increasing MPO expressionmay decrease anatabine accumulation, apparently by competitive use of acommon precursor in the formation of nicotine and other alkaloids.

Anabasine and anatalline contain a piperidine moiety. The piperidinemoiety of anabasine is thought to be derived from cadaverine viadelta-1-piperidine in tobacco. Watson A B, Brown, J. Chem. Soc. PerkinTrans. 1: 2607-2610 (1990). The conversion from cadaverine todelta-1-piperidine can be catalyzed by diamine oxidase Hashimoto et al.,Plant Physiol. 93: 216-221 (1990). Cadaverine is a good substrate forgeneral diamine oxidases but is also a substrate for MPO, although ithas a lower afffinity than N-methylated diamines. Hashimoto et al., id;Walton and McLauchlan, Phytochemistry 29: 1455-1457 (1990); Boswell etal., Phytochemistry 52: 871-878 (1999). The MPO genes of the presentinvention may therefore contribute to the formation of anabasine andanatalline.

Many other pyridyl bases plus many derivatives of nornicotine,anatabine, and anabasine are nicotinic alkaloids that have been reportedto be present in tobacco and for purposes of the invention shall beincluded within minor Nicotiana alkaloids or nicotinic alkaloids. Mostof these so-called “minor nicotinic alkaloids” are present in less than50 μg/g (dry weight basis) and many others are present in nanogramamounts. Bush, L. P., et al., “Biosynthesis and metabolism in nicotineand related alkaloids” in NICOTINE AND RELATED ALKALOIDS, J. W. Gorrod &J. Wahren (eds.) Chapman & Hall, London (1993); Bush, L. P., et al.,“Alkaloid Biosynthesis” in TOBACCO PRODUCTION CHEMISTRY AND TECHNOLOGY,.L. Davis and M. T. Nielson (eds.) Wiley, 1999.

“Nicotine” is the primary alkaloid in N. tabacum along with 50-60percent of other species of Nicotiana. Based on alkaloid accumulation inthe leaves, nornicotine, anatabine, and anabasine are the other foremostalkaloids in N. tabacum. Anatabine is usually not the primary alkaloidin any species but does accumulate to relatively higher amounts in 3species; anabasine is the primary alkaloid in four species. Nornicotineis the primary alkaloid in 30 to 40 percent of Nicotiana species.Depending on the variety, about 85 to about 95 percent of totalalkaloids in N. tabacum is nicotine. Bush, L. P., et al., “AlkaloidBiosynthesis” in Tobacco Production, Chemistry and Technology, D. L.Davis and M. T. Nielson (eds.) Wiley pp. 285-291 (1999); Hoffmann, etal., Journal of Toxicology and Environmental Health, 41:1-52, (1994).

A “reduced-nicotine plant” encompasses a plant that contains less thanhalf, preferably less than 25%, and more preferably less than 20% orless than 10% of the nicotine content of a control plant of the samevariety. A reduced-nicotine plant also includes a plant (Nicotiana,Duboisia, Solanum, Anthocercis and Salpiglessis genera in the Solanaceaeor the Eclipta and Zinnia genera in the Compositae) that contains lessnicotine compared with a control plant of the same variety.

A “reduced-alkaloid” plant encompasses a plant that contains less thanhalf, preferably less than 25%, and more preferably less than 20% orless than 10% of the “total alkaloid content” of a control plant of thesame variety.

A “reduced-anabasine” plant encompasses a plant that contains less thanhalf, preferably less than 25%, and more preferably less than 20% orless than 10% of the anabasine content of a control plant of the samevariety.

A “reduced-anatalline” plant encompasses a plant that contains less thanhalf, preferably less than 25%, and more preferably less than 20% orless than 10% of the anatalline content of control plant of the samevariety.

A “reduced-nornicotine” plant encompasses a plant that contains lessthan half, preferably less than 25%, and more preferably less than 20%or less than 10% of the anatalline content of a control plant of thesame variety.

A “reduced-anatabine” plant encompasses a plant that contains less thanhalf, preferably less than 25%, and more preferably less than 20% orless than 10% of the anatabine content of a control plant of the samevariety.

I. Nicotinic Alkaloid Reduction (Down-Regulation)

A. Decreasing Nicotiana Nicotinic Alkaloids by Suppressing at Least Oneof MPO1 and MPO2

Because MPO is a critical enzyme in the biosynthesis of nicotinicalkaloids, the inventive MPO sequences can be used for regulatingnicotinic alkaloids. That is, nicotinic alkaloid content may bedecreased by suppressing at least one of MPO1 and MPO2. Accordingly, thepresent invention provides methodology and constructs for decreasingnicotinic alkaloid content in a Nicotiana plant, by suppressing at leastone of MPO1 or MPO2. Suppressing both MPO1 and MPO2 may further decreasenicotinic alkaloids levels in a Nicotiana plant.

B. Decreasing Nicotiana Nicotinic Alkaloids by Suppressing at Least Oneof MPO1 and MPO2 and at Least One of A622, NBB1, QPT, and PMT

Previous reports indicate that suppressing more than one nicotinebiosynthesis gene in Nicotiana decreases nicotinic alkaloid contentfurther than suppressing one. For example, suppressing both A622 andNBB1 further reduces nicotine levels than suppressing either A622 orNBB1. See WO/2006/109197. Accordingly, the present inventioncontemplates further decreasing nicotinic alkaloid content bysuppressing at least one of MPO1 and MOP2 and one or more of A622, NBB1,QPT, and PMT in Nicotiana. Pursuant to this aspect of the invention, anucleic acid construct comprising a segment of at least one of MPO1 andMOP2 and one or more of A622, NBB1, QPT, and PMT is introduced into aNicotiana cell or plant. An illustrative nucleic acid construct maycomprise segments of both MPO1 and QPT.

II. Nicotinic Alkaloid Biosynthesis (Up-Regulation)

A. Increasing Nicotiana Nicotinic Alkaloids by Overexpressing at LeastOne of MPO1 and MPO2

The present invention also relates to increasing nicotinic alkaloids inNicotiana plants by overexpressing at least one of MPO1 and MPO2.Accordingly, the present invention provides methodology and constructsfor increasing nicotinic alkaloid content in a Nicotiana plant, byoverexpressing at least one of MPO1 and MPO2. Overexpressing at leastone of MPO1 and MPO2 to further increase nicotinic alkaloid levels in aNicotiana plant is similar to the methodology in Examples 7 and 8 of thepresent invention.

B. Increasing Nicotiana Nicotinic Alkaloids by Overexpressing at LeastOne of MPO1 and MPO2, and at Least One of PMT, QPT, A622, and NBB1

The only previous report demonstrating overexpression of a nicotinicbiosynthesis gene in any Nicotiana species was in N. sylvestris, wherePMT overexpression resulted in a modest 40% increase in leaf nicotine.Sato et al., Proc. Nat'l Acad. Sci. USA 98: 367-72 (2001). Whileoverexpressing a nicotinic alkaloid biosynthesis gene in one plantspecies, such as N. sylvestris, results in an increased accumulation ofsecondary metabolites, it does not necessarily follow that similaraccumulation of secondary metabolites will occur in a related species,such as N. tabacum. Saitoh et al., Phytochemistry 24: 477-80 (1985).This is especially relevant for PMT overexpression, since N. tabacumcontains five expressed PMT genes and N. sylvestris contains threeexpressed PMT genes. Hashimoto et al., Plant Mol. Biol. 37: 25-37(1998); Reichers & Timko, Plant Mol. Biol. 41: 387-401 (1999).

Indeed, when the PMT gene from N. tabacum was overexpressed in Duboisiahairy root cultures, the levels of nicotine, hyoscyamine, andscopolamine did not increase significantly. Moyano et al.,Phytochemistry 59, 697-702 (2002). Likewise, overexpressing the same PMTgene in transgenic plants and hairy root cultures of Atropa belladonnadid not affect hyoscyamine and scopolamine levels. Sato et al., Proc.Nat'l Acad. Sci. USA 98: 367-72 (2001); Rothe et al., J. Exp. Bot. 54:2065-070 (2003).

In Solanaceous species, such as tobacco, evidence suggests that the samealkaloid biosynthesis pathway in two related plant species can beregulated differently and overexpression of a given gene does notnecessarily lead to a similar accumulation pattern of secondarymetabolites. Moyano et al., J. Exp. Bot. 54: 203-11 (2003). For example,when sixty Nicotiana species were analyzed, there was considerablevariation in total alkaloid content and alkaloid profile amongst thespecies. Saitoh et al., Phytochemistry 24: 477-80 (1985).

For instance, while N. sylvestris had the highest dry weight content oftotal alkaloids (the sum of nicotine, nornicotine, anabasine andanatabine) at 29,600 pg/g or 2.96 percent, N. alata contained the lowestat 20 pg/g or 0.002 percent. The ratio of nicotine to total alkaloids inthe leaves of N. sylvestris was about 80 percent versus about 95 percentfor N. tabacum. Id. Also, the ratio of nornicotine to total alkaloid inN. sylvestris leaves was 19.1 percent versus 3 percent for N. tabacum

Based on these large variations among the sixty Nicotiana species,Saitoh et al. conclude that the “amount and ratio of total andindividual alkaloids present in a plant depend on the species. Noclear-cut correlation between alkaloid pattern and classification of thegenus Nicotiana seems to exist.” Id. at page 477.

Members of the Nicotiana genus can also differ in evolutionary originand other characteristics. See Clarkson et al., New Phytologist 168:241-252 (2005). For example, N. tabacum is an allotetraploid, has 24chromosomes, and is believed to originate from N. sylvestris (n=12) andN. tomentosiformis (n=12). N. benthamiana, indigenous to Australia, isalso an allotetraploid species, but it has 38 chromosomes and is thoughtto be the result of the hybridization of N. suaveolens (n=16) and N.debneyi (n=24). N. tabacum has about three fold the total alkaloidcontent of N. benthamiana and N. tabacum has a greater percentage of itstotal alkaloid content as nicotine. Saitoh et al., Phytochemistry 24:477-80 (1985).

It may be desirable, therefore, to overexpress different combinations ofnicotinic alkaloid biosynthesis genes in different species of Nicotianato produce elevated amounts of particular alkaloids. PMT, QPT, A622 andNBB1 up-regulation in N. tabacum increases nicotine biosynthesis.Nicotine can be further increased by genetically engineering more thanone gene in the nicotine biosynthesis pathway. See WO2007/072224.

Therefore, the present invention contemplates further increases ofnicotine synthesis in N. tabacum by overexpressing at least one of MPO1and MPO2, and at least one of A622, NBB1, QPT, and PMT. Pursuant to thisaspect of the invention, a nucleic acid construct comprising at leastone of MPO1 and MPO2, and at least one of A622, NBB1, QPT, and PMT isintroduced into a Nicotiana plant cell. An illustrative nucleic acidconstruct may comprise, for example, both MPO1 and PMT. See FIG. 6D.Similarly, for example, a genetically engineered plant overexpressingMPO1 and PMT may be produced by crossing a transgenic plantoverexpressing MPO1 with a transgenic plant overexpressing PMT.Following successive rounds of crossing and selection, a geneticallyengineered plant having overexpressing MPO1 and PMT can be selected.

C. Increasing Nicotiana Nicotinic Alkaloids and Yield

Increased nicotine plants of the invention may be produced byconventional breeding or crossing, as described by Wernsman et al., inPRINCIPLES OF CULTIVAR DEVELOPMENT—Vol. 2: CROP SPECIES (WR Fehr (ed.),Macrnillan, 1997). For example, a stable genetically engineeredtransformant, regenerated from tobacco material that contains a suitabletransgene, is employed to introgress a high-nicotine trait into adesirable commercially acceptable genetic background, thereby obtaininga tobacco cultivar or variety that combines a high nicotine level withthe desirable background.

While any desirable gene can be introgressed into a high-nicotinevariety, there is a critical need for introducing a high nicotine traitinto a high-yielding tobacco background. Several studies indicate that“Yield improvements have been hampered by the negative relationship thatexists with nicotine concentration.” (PRODUCTION, CHEMISTRY ANDTECHNOLOGY, D. L. Davis and M. T. Nielson (eds.) Wiley (1999), at page46). In his reflections of tobacco breeding, Wernsman asserts “continuedselection for yield alone will soon result in a population whosenicotine concentration in cured leaf is so low that the tobaccos areunacceptable to industry” Wernsman, Recent Advances in Tobacco Science25: 5-35 (1999). He postulates that “genetic methods of up-regulatingnicotine synthesis may be needed to permit additional increases inyielding ability while maintaining nicotine concentration” Id.

Accordingly, the present invention provides a means for correcting the“negative correlation” between yield and nicotine content in Nicotianaplants by overexpressing a gene encoding a nicotine biosynthesis enzymein a high-yielding Nicotiana plant. Exemplary nicotine biosynthesisenzymes include but are not limited to MPO1, MPO2, QPT, PMT, A622, NBB1,arginine decarboxylase (ADC), NADH dehydrogenase, ornithinedecarboxylase (ODC), and S-adenosyl-methionine synthetase (SAMS).Increased-nicotine plants resulting therefrom are then crossed with anydesirable commercially acceptable genetic background that maintains highyield. Suitable high-yield Nicotiana plants include but are not limitedto Nicotiana tabacum cultivars K326, NC71, NC72 and RG81. Followingsuccessive rounds of crossing and selection, a genetically engineeredplant having increased nicotine and increased yield is accordinglyproduced.

A further aspect of the invention provides crossing anincreased-nicotine plant with an increased-yield plant as anotherstrategy for breaking the negative correlation between nicotine contentand yield.

“Increased yield genes” encompass any gene whose expression correlateswith increased production capacity as reflected by, for example,increased photoassimilate production, increased growth rate, improvedvigor, enhanced yield, enhanced CO₂ fixation, enhanced biomass,increased seed production, improved storage, enhanced yield, increaseddisease tolerance, increased insect tolerance, increased water-stresstolerance, enhanced sweetness, improved starch composition, improvedsucrose accumulation and export, and improved response to oxidativestress compared with a wild-type control plant.

Likewise, an “increased yield plant” refers to a plant, or any portionthereof, overexpressing an “increased yield gene” and exhibits increasedproduction capacity as reflected by, for example, increasedphotoassimilate production, increased growth rate, improved vigor,enhanced yield, enhanced CO₂ fixation, enhanced biomass, increased seedproduction, improved storage, enhanced yield, increased diseasetolerance, increased insect tolerance, increased water-stress tolerance,enhanced sweetness, improved starch composition, improved sucroseaccumulation and export, and improved response to oxidative stresscompared with a wild-type control plant.

For example, and in no way limiting the invention, an increased yieldplant can be produced by overexpressing a pathogenesis-related (PR)gene. It has been shown that overexpressing a maize PRms gene, intobacco produced transgenic tobacco plants having enhanced biomass andseed production. See Murillo et al., Plant J 36: 330-41 (2003), which isincorporated herein by reference. Likewise, an increased yield plant canbe produced by overexpressing a gene encoding a Calvin cycle enzyme. SeeTamoi et al. Plant Cell Physiol. 47(3)3 80-390 (2006), which isincorporated herein by reference. Tobacco plants overexpressing, forexample, a cyanobacterial fructose-1,6-/sedoheptulose-1,7-bisphosphatasedisplayed enhanced photosynthetic efficiency and growth efficiencycompared with wild-type tobacco. See Miyagawa et al., Nature Biotech.19: 965-69 (2001).

The present invention also contemplates producing a plant havingincreased yield and increased nicotine by overexpressing a gene encodinga nicotine biosynthesis enzyme, such as MPO1, MPO2, QPT, PMT, A622, orNBB1, and overexpressing an increased yield gene, such as genes encodingPRms, fructose-1,6-/sedoheptulose-1,7-bisphosphatase,fructose-1,6-bisphosphatase, and sedoheptulose-1,7-bisphosphatase,sedoheptulose-1,7-bisphosphatase in the same plant or cell.

D. Producing Nicotinic Alkaloids and Related Compounds in Non-NicotineProducing Cells

At least one of MPO1 and MPO2 can be introduced into a non-nicotineproducing plant or cell, thereby producing nicotine or related compoundsin an organism or cell that does not produce these compounds otherwise.A variety of products can be produced from these engineered organismsand cells, including nicotine, nicotine precursors, nicotine analogs,and nicotine biosynthesis enzymes.

A “non-nicotine producing plant” refers to any plant that does notproduce nicotine or related nicotinic alkaloids. Illustrativenon-nicotine producing plants include but are not limited to Atropabelladonna and Arabidopsis thaliana.

“Non-nicotine producing cells” refers to cells from any organism thatdoes not produce nicotine or related nicotinic alkaloids. Illustrativecells include but are not limited to plant cells, such as Atropabelladonna, Arabidopsis thaliana, as well as insect, mammalian, yeast,fungal, algal, or bacterial cells.

A “nicotine analog” has the basic structure of nicotine but may, forexample, have different ring substituents. For example, a nicotineanalog may substitute a hydrogen (—H) for the methyl group (—CH3)thereby producing nornicotine, which is an analog of nicotine. Inaddition to sharing a similar structure with nicotine, nicotine analogsmay provide similar physiological effects. Cotinine, for example, hasbeen cited for its positive effects on improving concentration andmemory and, accordingly, is a nicotine analog. Accordingly, nicotineanalogs are defined broadly to cover any and all compounds havingsimilar structural and functional activity to nicotine.

III. Nicotinic Alkaloid Ratio Regulation

In addition to (a) suppressing nicotine biosynthesis gene(s) to producereduced-nicotine tobacco plants and products or (b) up-regulatingnicotine biosynthesis gene(s) to produce increased-nicotine tobaccoplants and products, an object of the present invention is to alter theratio of nicotine-to-total alkaloid content of plants, N. tabacum plantsbeing preferred, thus changing the alkaloid profile of a plant. Thetotal alkaloid content may be approximately the same, or may be higheror lower.

For example, nicotine and anatabine are formed by the addition ofdifferent heterocyclic rings to the same pyridine precursor derived fromnicotinic acid. See FIG. 1A. Increasing the level of N-methylpyrroliniumion increases the ratio of the nicotine-specific precursorN-methylpyrrolinium ion to the common pyridine precursor and also theratio of the N-methylpyrrolinium ion to the anatabine specific precursor3,6-dihydropyridine, resulting in an increase in the ratio of nicotineto anatabine. The level of N-methylpyrrolinium ion can be increased byupregulating at least one of MPO1, MPO2 and PMT.

Additionally, the ratio of N-methylpyrrolinium ion to the commonpyridine precursor may be further increased by suppressing a geneencoding an enzyme required for synthesis of nicotinic acid, such asQPT. For example, since the specific precursor of anatabine,3,6-dhydropyridine, is also derived from nicotinic acid this will alsoincrease the ratio of the nicotine-specific precursor to theanatabine-specific precursor.

As pertaining to traditional tobacco products such as snus, an advantageof genetically engineering a tobacco plant's alkaloid profile in whichnicotinic alkaloids levels other than nicotine are reduced is thatcertain TSNAs, such as N′-nitrosoanatabine (NAT) and N′-nitrosoanabasine(NAB) are reduced. Xie et al. (2004); Djordjevic et al., J. Agric. FoodChem., 37: 752-756 (1989).

IV. Products

A. Reduced Nicotine Products

Reducing total alkaloid content in tobacco would increase the value oftobacco as a biomass resource. When grown under conditions that maximizebiomass, such as high density and multiple cuttings, tobacco can yieldmore than 8 tons dry weight per acre, which is comparable with othercrops used for biomass. Large-scale growing and processing ofconventional tobacco biomass has several drawbacks, however. Forexample, significant time and energy is spent extracting, isolating, anddisposing tobacco alkaloids because conventional tobacco biomass,depending on the variety, contains about 1 to about 5 percent alkaloids.On a per acre basis, conventional tobacco biomass contains approximatelyas much as 800 pounds of alkaloids. Also, people handling tobacco maysuffer from overexposure to nicotine, commonly referred to as “greentobacco disease.”

An additional use of reduced-nicotine tobacco is for smoking cessation.More successful methods are needed to assist smokers in quittingsmoking. Nicotine replacement therapy (NRT) is not very effective as asmoking cessation treatment because its success rate is less than 20percent after 6 to 12-months from the end of the nicotine replacementperiod. Bohadana et al., Arch Intern. Med. 160:3 128-3 134 (2000);Croghan et al., Nicotine Tobacco Res. 5: 18 1-1 87 (2003); Stapleton etal., Addiction 90:3 1-42 (1995). Nicotine-reduced or nicotine-freetobacco cigarettes have assisted smokers in quitting smokingsuccessfully, by weaning the smoker from nicotine yet allowing thesmoker to perform the smoking ritual. Additionally, denicotinizedcigarettes relieve craving and other smoking withdrawal symptoms. SeeRose, Psychopharmacology 184: 274-285 (2006) and Rose et al., NicotineTobacco Res. 8:89-101 (2006).

B. Increased Nicotine Products

In addition to the more traditional applications for increased nicotineproducts, such as cigarettes and other tobacco products, recentpharmacological studies suggest a therapeutic role for nicotine andrelated compounds. For example, several research groups are presentlystudying drugs that target nicotine receptors as a means for treatingcognitive impairments, such as Alzheimer's disease, schizophrenia, andage-related memory loss. Singer, E., “The Upside to Nicotine,”Technology Review (Jul. 28, 2006). Acetylcholine receptor ligands, suchas nicotine, have been demonstrated to have effects on attention,cognition, appetite, substance abuse, memory, extra pyramidal function,cardiovascular function, pain, and gastrointestinal motility andfunction. U.S. Pat. No. 5,852,041. Accordingly, there are therapeuticbenefits of nicotine and related compounds, and thus there is a need forimproved methods for producing them.

V. Synthesis of Compounds Using Novel Enzymes

Recently, there has been great interest in synthesizing nicotine analogsthat target nicotine receptors and provide therapeutic effects forneurogenerative diseases and cognitive disabilities. For example,Targacept, a pharmaceutical company formed as a spinout from R.J.Reynolds Tobacco Company, endeavors to develop and commercializenicotine analog drugs based on selective activation of neuronalnicotinic acetylcholine receptors (NNRs). The present invention providesnovel nucleic acids encoding nicotine biosynthesis enzymes, which may bevaluable in using at least one of MPO1 and MPO2, or at least two ofMPO1, MPO2, A622, NBB1, QPT, or PMT, for developing novel nicotineanalogs. For example, using the inventive methods and constructs, anicotinic alkaloid analog can be produced by providing a nicotine analogprecursor in a cell culture system comprising cells overexpressing atleast one of MPO1 and MPO2.

Additionally, the enzymes produced from the inventive nucleic acidsequences may be used for in vitro synthesis of nicotine and relatedcompounds. That is, recombinant MPO1 and MPO2 can be used for thesynthesis or partial synthesis of a nicotinic alkaloid and a nicotinicalkaloid analog.

The enzymes produced from the inventive nucleic acid sequences may beused for synthesizing alkaloids other than nicotinic alkaloids,including tropane alkaloids, as well as precursors of such alkaloids,and analogs of such alkaloids and alkaloid precursors.

MPO enzymes are known to accept, to varying degrees, substrates otherthan N-methylputrescine. Hashimoto et al., Plant Physiol. 93: 216(1990); Walton et al., Phytochemistry 29: 1455 (1990); McLauchlan etal., Planta 191: 440 (1993); Boswell et al., Phytochemistry 52: 855(1999).

Nicotinic Alkaloid Biosynthesis Sequences

Nicotinic alkaloid biosynthesis genes have been identified in severalplant species, exemplified by Nicotiana plants. Accordingly, the presentinvention embraces any nucleic acid, gene, polynucleotide, DNA, RNA,mRNA, or cDNA molecule that is isolated from the genome of a plantspecies, or produced synthetically, that increases Nicotiana nicotinicalkaloid biosynthesis. Additionally, expression of such nicotinicalkaloid biosynthesis sequence produces nicotinic alkaloids in anon-nicotine producing cell, such as an insect cell. The DNA or RNA maybe double-stranded or single-stranded. Single-stranded DNA may be thecoding strand, also known as the sense strand, or it may be thenon-coding strand, also called the antisense strand.

It is understood that the terms MPO1 and MPO2 designate the sequencesset forth in SEQ ID NO: 1 and 3, respectively, as well as nucleic acidmolecules comprised of variants of SEQ ID NO: 1 and 3, with one or morebases deleted, substituted, inserted, or added, which variant codes fora polypeptide with nicotinic alkaloid biosynthesis activity.Accordingly, sequences having “base sequences with one or more basesdeleted, substituted, inserted, or added” retain physiological activityeven when the encoded amino acid sequence has one or more amino acidssubstituted, deleted, inserted, or added. Additionally, multiple formsof MPO1 and MPO2 may exist, which may be due to posttranslationalmodification of a gene product, or to multiple forms of the respectiveMPO1 or MPO2, genes. Nucleotide sequences that have such modificationsand that code for a nicotinic alkaloid biosynthesis enzyme are includedwithin the scope of the present invention.

For example, the poly A tail or 5′- or 3′-end, nontranslation regionsmay be deleted, and bases may be deleted to the extent that amino acidsare deleted. Bases may also be substituted, as long as no frame shiftresults. Bases also may be “added” to the extent that amino acids areadded. It is essential, however, that any such modification does notresult in the loss of nicotinic alkaloid biosynthesis enzyme activity. Amodified DNA in this context can be obtained by modifying the DNA basesequences of the invention so that amino acids at specific sites aresubstituted, deleted, inserted, or added by site-specific mutagenesis,for example. Zoller & Smith, Nucleic Acid Res. 10: 6487-500 (1982).

A nicotinic alkaloid biosynthesis sequence can be synthesized ab initiofrom the appropriate bases, for example, by using an appropriate proteinsequence disclosed herein as a guide to create a DNA molecule that,though different from the native DNA sequence, results in the productionof a protein with the same or similar amino acid sequence. This type ofsynthetic DNA molecule is useful when introducing a DNA sequence into anon-plant cell, coding for a heterologous protein, that reflectsdifferent (non-plant) codon usage frequencies and, if used unmodified,can result in inefficient translation by the host cell.

By “isolated” nucleic acid molecule(s) is intended a nucleic acidmolecule, DNA or RNA, which has been removed from its nativeenvironment. For example, recombinant DNA molecules contained in a DNAconstruct are considered isolated for the purposes of the presentinvention. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells or DNAmolecules that are purified, partially or substantially, in solution.Isolated RNA molecules include in vitro RNA transcripts of the DNAmolecules of the present invention. Isolated nucleic acid molecules,according to the present invention, further include such moleculesproduced synthetically.

“Exogenous nucleic acid” refers to a nucleic acid, DNA or RNA, which hasbeen introduced into a cell (or the cell's ancestor) through the effortsof humans. Such exogenous nucleic acid may be a copy of a sequence whichis naturally found in the cell into which it was introduced, orfragments thereof.

In contrast, “endogenous nucleic acid” refers to a nucleic acid, gene,polynucleotide, DNA, RNA, rnRNA, or cDNA molecule that is present in thegenome of a plant or organism that is to be genetically engineered. Anendogenous sequence is “native” to, i.e., indigenous to, the plant ororganism that is to be genetically engineered.

“Heterologous nucleic acid” refers to a nucleic acid, DNA or RNA, whichhas been introduced into a cell (or the cell's ancestor) which is not acopy of a sequence naturally found in the cell into which it isintroduced. Such heterologous nucleic acid may comprise segments thatare a copy of a sequence which is naturally found in the cell into whichit has been introduced, or fragments thereof.

A “chimeric nucleic acid” comprises a coding sequence or fragmentthereof linked to a transcription initiation region that is differentfrom the transcription initiation region with which it is associated incells in which the coding sequence occurs naturally.

Unless otherwise indicated, all nucleotide sequences determined bysequencing a DNA molecule herein were determined using an automated DNAsequencer, such as the Model 373 from Applied Biosystems, Inc.Therefore, as is known in the art for any DNA sequence determined bythis automated approach, any nucleotide sequence determined herein maycontain some errors. Nucleotide sequences determined by automation aretypically at least about 95% identical, more typically at least about96% to at least about 99.9% identical to the actual nucleotide sequenceof the sequenced DNA molecule. The actual sequence can be more preciselydetermined by other approaches including manual DNA sequencing methodswell known in the art. As is also known in the art, a single insertionor deletion in a determined nucleotide sequence compared to the actualsequence will cause a frame shift in translation of the nucleotidesequence such that the predicted amino acid sequence encoded by adetermined nucleotide sequence may be completely different from theamino acid sequence actually encoded by the sequenced DNA molecule,beginning at the point of such an insertion or deletion.

For the purpose of the invention, two sequences hybridize when they forma double-stranded complex in a hybridization solution of 6×SSC, 0.5%SDS, 5×Denhardt's solution and 100 pg of non-specific carrier DNA. SeeAusubel et al., supra, at section 2.9, supplement 27 (1994). Sequencesmay hybridize at “moderate stringency,” which is defined as atemperature of 60° C. in a hybridization solution of 6×SSC, 0.5% SDS,5×Denhardt's solution and 100 μg of non-specific carrier DNA. For “highstringency” hybridization, the temperature is increased to 68° C.Following the moderate stringency hybridization reaction, thenucleotides are washed in a solution of 2×SSC plus 0.05% SDS for fivetimes at room temperature, with subsequent washes with 0.1×SSC plus 0.1%SDS at 60° C. for 1 h. For high stringency, the wash temperature isincreased to 68° C. For the purpose of the invention, hybridizednucleotides are those that are detected using 1 ng of a radiolabeledprobe having a specific radioactivity of 10,000 cpm/ng, where thehybridized nucleotides are clearly visible following exposure to X-rayfilm at −70° C. for no more than 72 hours.

“Sequence identity” or “identity” in the context of two nucleic acid orpolypeptide sequences includes reference to the residues in the twosequences which are the same when aligned for maximum correspondenceover a specified region. When percentage of sequence identity is used inreference to proteins it is recognized that residue positions which arenot identical often differ by conservative amino acid substitutions,where amino acid residues are substituted for other amino acid residueswith similar chemical properties, such as charge and hydrophobicity, andtherefore do not change the functional properties of the molecule. Wheresequences differ in conservative substitutions, the percent sequenceidentity may be adjusted upwards to correct for the conservative natureof the substitution. Sequences which differ by such conservativesubstitutions are said to have “sequence similarity” or “similarity.”Means for making this adjustment are well-known to those of skill in theart. Typically this involves scoring a conservative substitution as apartial rather than a full mismatch, thereby increasing the percentagesequence identity. Thus, for example, where an identical amino acid isgiven a score of 1 and a non-conservative substitution is given a scoreof zero, a conservative substitution is given a score between zeroand 1. The scoring of conservative substitutions is calculated, forexample, according to the algorithm of Meyers & Miller, Computer Applic.Biol. Sci. 4: 11-17 (1988), as implemented in the program PC/GENE(Intelligenetics, Mountain View, Calif., USA).

Use in this description of a percentage of sequence identity denotes avalue determined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

The present application is directed to such nucleic acid molecules whichare at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%,or 100% identical to a nucleic acid sequence described in any of SEQ IDNO: 1, 3. Preferred are nucleic acid molecules which are at least 95%,96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence shownin any of SEQ ID NO: 1, 3. Differences between two nucleic acidsequences may occur at the 5′ or 3′ terminal positions of the referencenucleotide sequence or anywhere between those terminal positions,interspersed either individually among nucleotides in the referencesequence or in one or more contiguous groups within the referencesequence.

As a practical matter, whether any particular nucleic acid molecule isat least 95%, 96%, 97%, 98%, or 99% identical to a reference nucleotidesequence refers to a comparison made between two molecules usingstandard algorithms well known in the art and can be determinedconventionally using publicly available computer programs such as theBLASTN algorithm. See Altschul et al., Nucleic Acids Res. 25: 3389-402(1997).

The present invention further provides nucleic acid molecules comprisingthe nucleotide sequence of SEQ ID NOS.: 1,3, respectively, which encodean active nicotine biosynthesis MPO enzyme, wherein the enzyme has aminoacid sequence that corresponds to SEQ ID NO.: 2 and 4, respectively, andwherein the protein of the invention encompasses amino acidsubstitutions, additions and deletions that do not alter the function ofthe nicotine biosynthesis enzyme.

A “variant” is a nucleotide or amino acid sequence that deviates fromthe standard, or given, nucleotide or amino acid sequence of aparticular gene or protein. The terms “isoform,” “isotype,” and “analog”also refer to “variant” forms of a nucleotide or an amino acid sequence.An amino acid sequence that is altered by the addition, removal, orsubstitution of one or more amino acids, or a change in nucleotidesequence, may be considered a “variant” sequence. The variant may have“conservative” changes, wherein a substituted amino acid has similarstructural or chemical properties, e.g., replacement of leucine withisoleucine. A variant may have “nonconservative” changes, e.g.,replacement of a glycine with a tryptophan. Analogous minor variationsmay also include amino acid deletions or insertions, or both. Guidancein determining which amino acid residues may be substituted, inserted,or deleted may be found using computer programs well known in the artsuch as Vector NTI Suite (InforMax, MD) software. “Variant” may alsorefer to a “shuffled gene” such as those described in Maxygen-assignedpatents.

Methodology for Reducing Alkaloids

In one aspect of the invention, methods are provided for reducingalkaloid levels. While any method may be used for reducing alkaloidlevels, the present invention contemplates antisense, senseco-suppression, RNAi, artificial microRNA, ribozyme, and virus-inducedgene silencing (VIGS), and targeted mutagenesis approaches.

For example, a heterologous sequence utilized in the antisense methodsof the present invention may be selected so as to produce an RNA productcomplementary to an entire MPO1 or MPO2 mRNA sequence, or to a portionthereof. The sequence may be complementary to any contiguous sequence ofthe natural messenger RNA, that is, it may be complementary to theendogenous mRNA sequence proximal to the 5′-terminus or capping site,downstream from the capping site, between the capping site and theinitiation codon and may cover all or only a portion of the non-codingregion, may bridge the non-coding and coding region, be complementary toall or part of the coding region, complementary to the 3′-terminus ofthe coding region, or complementary to the 3′-untranslated region of themRNA.

Suitable antisense sequences may be from at least about 13 to about 15nucleotides, at least about 16 to about 21 nucleotides, at least about20 nucleotides, at least about 30 nucleotides, at least about 50nucleotides, at least about 75 nucleotides, at least about 100nucleotides, at least about 125 nucleotides, at least about 150nucleotides, at least about 200 nucleotides, or more. In addition, thesequences may be extended or shortened on the 3′ or 5′ ends thereof.

The particular antisense sequence and the length of the antisensesequence will vary, depending, for example, upon the degree ofinhibition desired and the stability of the antisense sequence.Generally available techniques and the information provided in thisspecification can guide the selection of appropriate MPO1 or MPO2antisense sequences. With reference to SEQ ID NO: 1 or 3 herein, anoligonucleotide of the invention may be a continuous fragment of MPO1 orMPO2 cDNA sequence in antisense orientation, of any length that issufficient to achieve the desired effects when transformed into arecipient plant cell.

The present invention may contemplate sense co-suppression of one orboth of MPO1 and MPO2. Sense polynucleotides employed in carrying outthe present invention are of a length sufficient to suppress, whenexpressed in a plant cell, the native expression of the plant MPO1 orMPO2 protein in that plant cell. Such sense polynucleotides may beessentially an entire genomic or complementary nucleic acid encoding theMPO1 or MPO2 enzyme, or a fragment thereof, with such fragmentstypically being at least 15 nucleotides in length. Techniques aregenerally available for ascertaining the length of sense DNA thatresults in suppression of the expression of a native gene in a cell.

In an alternate embodiment of the present invention, plant cells aretransformed with a nucleic acid construct containing a polynucleotidesegment encoding an enzymatic RNA molecule (a “ribozyme”), whichenzymatic RNA molecule is directed against (i.e., cleaves) the mRNAtranscript of DNA encoding MPO1 or MPO2, as described herein. Ribozymescontain substrate binding domains that bind to accessible regions of thetarget mRNA, and domains that catalyze the cleavage of RNA, preventingtranslation and protein production. The binding domains may compriseantisense sequences complementary to the target mRNA sequence; thecatalytic motif may be a hammerhead motif or other motifs, such as thehairpin motif.

Ribozyme cleavage sites within an RNA target may initially be identifiedby scanning the target molecule for ribozyme cleavage sites (e.g., GUA,GUU or GUC sequences). Once identified, short RNA sequences of 15, 20,30, or more ribonucleotides corresponding to the region of the targetgene containing the cleavage site may be evaluated for predictedstructural features.

The suitability of candidate targets also may be evaluated by testingtheir accessibility to hybridization with complimentaryoligonucleotides, using ribonuclease protection assays as are known inthe art. DNA encoding enzymatic RNA molecules may be produced inaccordance with known techniques. For example, see Cech et al., U.S.Pat. No. 4,987,071; Keene et al., U.S. Pat. No. 5,559,021; Donson etal., U.S. Pat. No. 5,589,367; Torrence et al., U.S. Pat. No. 5,583,032;Joyce, U.S. Pat. No. 5,580,967; Gold et al., U.S. Pat. No. 5,595,877;Wagner et al., U.S. Pat. No. 5,591,601; and U.S. Pat. No. 5,622,854.

Production of such an enzymatic RNA molecule in a plant cell anddisruption of MPO1 or MPO2 protein production reduces protein activityin plant cells, in essentially the same manner as production of anantisense RNA molecule; that is, by disrupting translation of mRNA inthe cell which produces the enzyme. The term “ribozyme” describes anRNA-containing nucleic acid that functions as an enzyme, such as anendoribonuclease, and may be used interchangeably with “enzymatic RNAmolecule.”

The present invention further includes nucleic acids encoding ribozymes,nucleic acids that encode ribozymes and that have been inserted into anexpression vector, host cells containing such vectors, and methodologyemploying ribozymes to decrease MPO1 and MPO2 expression in plants.

In one embodiment, the present invention provides double-strandednucleic acid molecules of that mediate RNA interference gene silencing.In another embodiment, the siNA molecules of the invention consist ofduplex nucleic acid molecules containing about 15 to about 30 base pairsbetween oligonucleotides comprising about 15 to about 30 (e.g., about15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30)nucleotides. In yet another embodiment, siNA molecules of the inventioncomprise duplex nucleic acid molecules with overhanging ends of about 1to about 32 (e.g., about 1,2, or 3) nucleotides, for example, about21-nucleotide duplexes with about 19 base pairs and 3′-terminalmononucleotide, dinucleotide, or trinucleotide overhangs. In yet anotherembodiment, siNA molecules of the invention comprise duplex nucleic acidmolecules with blunt ends, where both ends are blunt, or alternatively,where one of the ends is blunt.

An siNA molecule of the present invention may comprise modifiednucleotides while maintaining the ability to mediate RNAi. The modifiednucleotides can be used to improve in vitro or in vivo characteristicssuch as stability, activity, and/or bioavailability. For example, a siNAmolecule of the invention can comprise modified nucleotides as apercentage of the total number of nucleotides present in the siNAmolecule. As such, a siNA molecule of the invention can generallycomprise about 5% to about 100% modified nucleotides (e.g., about 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95% or 100% modified nucleotides). The actual percentageof modified nucleotides present in a given siNA molecule will depend onthe total number of nucleotides present in the siNA. If the siNAmolecule is single stranded, the percent modification can be based uponthe total number of nucleotides present in the single stranded siNAmolecules. Likewise, if the siNA molecule is double stranded, thepercent modification can be based upon the total number of nucleotidespresent in the sense strand, antisense strand, or both the sense andantisense strands.

The present invention includes nucleic acids encoding comprising RNAigene silencing vectors, host cells containing such vectors, andmethodology employing RNAi vectors to decrease MPO1 and MPO2 expressionin plants. RNAi constructs generate siNA in situ from transcripts thatcontain segments of target sequences in an inverted repeat orientation,allowing formation of “hairpin” RNAs containing double-stranded regions.Reviews on the design and use of such RNAi constructs include McGinniset al. Methods Enzymol. 392: 1-24 (2005), Watson et al. (FEBS Lett.579:5982-7 (2005), Wesley et al., Methods Mol. Biol. 236:273-86 (2003).RNAi constructs containing segments of target gene sequences rangingfrom less than 100 nt to more than 800 nt are useful in suppressinggenes in plants. Wesley et al., Plant J. 27: 581-590 (2001).

Artificial microRNAs, which have double stranded regions comprisingtarget gene sequences of only about 21 bases, are effective forsuppression of gene expression in plants. Schwab et al., Plant Cell 18:1121-1133 (2006), Lu et al., Nucleic Acids Research 32: e171 (2004)Nucleic acids comprising segments of the MPO1 and MPO2 that are distinctor identical may be incorporated in artificial microRNAs for suppressionof MPO1, MPO2, or both MPO1 and MPO2.

The present invention further includes nucleic acids encoding comprisingvirus-induced gene silencing (VIGS) vectors, host cells containing suchvectors, and methodology employing VIGS vectors to decrease MPO1 andMPO2 expression in plants. Liu et al. The Plant Journal 30(4), 415-429(2002).

In one embodiment, the present invention provides VIGS vectors whichcomprise nucleic acid molecules containing MPO1 or MPO2oligonucleotides. The MPO1 or MPO2 oligonucleotides may be about 15 toabout 30 bases (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30) nucleotides or longer.

Expression may be reduced by introducing a nucleic acid comprising aportion of an MPO1 or MPO2 sequence that causes targeted in situmutagenesis of an endogenous gene, resulting in its inactivation.

The MPO1 and MPO2 sequences of the present invention can be used inmethods for screening for mutations in specific target gene regions. Forexample, specific primers comprising segments of the MPO1 or MPO2sequences are used to amplify a region of the corresponding gene in DNAfrom pools of mutagenized plants, and the presence of mutations withinthe segment is detected by cleavage of heteroduplexes formed by mutantand wild type sequences (Till et al. BMC Plant Biol. 7: 19 (2007)). Thisapproach was used to identify mutations in nicotine demethylase. U.S.published patent application 2007/0199097. Alternatively, mutations canbe identified by sequencing of the DNAs amplified using the specificprimers, or direct sequencing of the DNA from mutagenized plants usingprimers comprising segments of the MPO1 and MPO2 nucleic acids of thepresent invention. Mutations in MPO1 and MPO2 can also be detected byhybridizing nucleic acids comprising nucleic acids of the presentinvention to probes comprising regions of the corresponding genes frommutagenized plants arrayed on microarrays and observing differentialhybridization.

The MPO1 and MPO2 sequences of the present invention can be used inoligonucleotide-directed gene repair, in which oligonucleotides, whichmay be chimeric nucleotides composed of DNA and modified RNA residues,are used to induce mutations at specific target sites. Theoligonucleotide mutational vectors may include substitutions of U for Tand substitions of non-natural nucleotide analogs. Zhu et al., ProcNat'l Acad Sci USA 96: 8768-73 (1999), Beetham et al., Proc Nat'l AcadSci USA 96: 8774-8 (1999); Kipp et al. Methods Mol Biol 133: 213-21(2000); Dong et al., Plant Cell Rep. 25: 457-65 (2006); U.S. Pat. Nos.6,271,360, 6,479,292, and 7,060,500.

Nucleic Acid Constructs

In accordance with one aspect of the invention, a sequence thatsuppresses nicotinic alkaloid biosynthesis is incorporated into anucleic acid construct that is suitable for plant or celltransformation. Thus, such a nucleic acid construct can be used tosuppress at least one of MPO1 and MPO2. Further, a nucleic acidconstruct at can be used to suppress at least one of MPO1 and MPO2 and,in addition, at least one of A622, NBB1, PMT, and QPT in a plant.

In another aspect of the invention, a sequence that increases nicotinicalkaloid biosynthesis is incorporated into a nucleic acid construct thatis suitable for plant or cell transformation. Thus, such a nucleic acidconstruct can be used to overexpress at least one of MPO1 and MPO2 in aplant. Further, such a nucleic acid construct can be used to overexpressat least one of MPO1 and MPO2, and, in addition, at least one of A622and NBB1, PMT and QPT in a plant, or in a non-nicotine producing cell.

Recombinant nucleic acid constructs may be made using standardtechniques. For example, the DNA sequence for transcription may beobtained by treating a vector containing said sequence with restrictionenzymes to cut out the appropriate segment. The DNA sequence fortranscription may also be generated by annealing and ligating syntheticoligonucleotides or by using synthetic oligonucleotides in a polymerasechain reaction (PCR) to give suitable restriction sites at each end. TheDNA sequence then is cloned into a vector containing suitable regulatoryelements, such as upstream promoter and downstream terminator sequences.

An important aspect of the present invention is the use of nucleic acidconstructs wherein a nicotinic alkaloid biosynthesis-encoding sequenceis operably linked to one or more regulatory sequences, which driveexpression of the nicotinic alkaloid biosynthesis-encoding sequence incertain cell types, organs, or tissues without unduly affecting normaldevelopment or physiology.

“Promoter” connotes a region of DNA upstream from the start oftranscription that is involved in recognition and binding of RNApolymerase and other proteins to initiate transcription. A “constitutivepromoter” is one that is active throughout the life of the plant andunder most environmental conditions. Tissue-specific, tissue-preferred,cell type-specific, and inducible promoters constitute the class of“non-constitutive promoters.” “Operably linked” refers to a functionallinkage between a promoter and a second sequence, where the promotersequence initiates and mediates transcription of the DNA sequencecorresponding to the second sequence. In general, “operably linked”means that the nucleic acid sequences being linked are contiguous.

Promoters useful for expression of a nucleic acid sequence introducedinto a cell to either decrease or increase expression of MPO1, MPO2,A622, NBB1, PMT, or QPT may be constitutive promoters, such as thecauliflower mosaic virus (CaMV) 35S promoter, or tissuespecific,tissue-preferred, cell type-specific, and inducible promoters. Preferredpromoters include promoters which are active in root tissues, such asthe tobacco RB7 promoter (Hsu et al. Pestic. Sci. 44: 9-19 (1995); U.S.Pat. No. 5,459,252), maize promoter CRWAQ81 (US published patentapplication 20050097633); the Arabidopsis ARSK1 promoter (Hwang andGoodman, Plant J 8:37-43 (1995)), the maize MR7 promoter (U.S. Pat. No.5,837,848), the maize ZRP2 promoter (U.S. Pat. No. 5,633,363), the maizeMTL promoter (U.S. Pat. Nos. 5,466,785 and 6,018,099) the maize MRS1,MRS2, MRS3, and MRS4 promoters (U.S. Pat. App. 200500 10974), anArabidopsis cryptic promoter (U.S. Pat. App. 20030106105) and promotersthat are activated under conditions that result in elevated expressionof enzymes involved in nicotine biosynthesis such as the tobacco RD2promoter (U.S. Pat. No. 5,837,876), PMT promoters (Shoji T. et al.,Plant Cell Physiol. 41: 831-39 (2000b); WO 2002/038588) or an A622promoter (Shoji T. et al., Plant Mol. Biol. 50: 427-40 (2002)).

The vectors of the invention may also contain termination sequences,which are positioned downstream of the nucleic acid molecules of theinvention, such that transcription of mRNA is terminated, and polyAsequences added. Exemplary of such terminators are the cauliflowermosaic virus (CaMV) 35S terminator and the nopaline synthase gene (Tnos)terminator. The expression vector also may contain enhancers, startcodons, splicing signal sequences, and targeting sequences.

Expression vectors of the invention may also contain a selection markerby which transformed cells can be identified in culture. The marker maybe associated with the heterologous nucleic acid molecule, i.e., thegene operably linked to a promoter. As used herein, the term “marker”refers to a gene encoding a trait or a phenotype that permits theselection of, or the screening for, a plant or cell containing themarker. In plants, for example, the marker gene will encode antibioticor herbicide resistance. This allows for selection of transformed cellsfrom among cells that are not transformed or transfected.

Examples of suitable selectable markers include adenosine deaminase,dihydrofolate reductase, hygromycin-B-phosphotransferase, thyrnidnekinase, xanthine-guanine phospho-ribosyltransferase, glyphosate andglufosinate resistance, and amino-glycoside 3′-O-phosphotranserase(kanamycin, neomycin and G418 resistance). These markers may includeresistance to G418, hygromycin, bleomycin, kanamycin, and gentamicin.The construct may also contain the selectable marker gene bar thatconfers resistance to phosphinothricin (glufosinate) and bialafos.Thompson et al., EMBO J. 9: 2519-23 (1987). Other suitable selectionmarkers are known as well.

Visible markers such as green florescent protein (GFP) may be used.Methods for identifying or selecting transformed plants based on thecontrol of cell division have also been described. See WO 2000/052168and WO 2001/059086.

Replication sequences, of bacterial or viral origin, may also beincluded to allow the vector to be cloned in a bacterial or phage host.Preferably, a broad host range prokaryotic origin of replication isused. A selectable marker for bacteria may be included to allowselection of bacterial cells bearing the desired construct. Suitableprokaryotic selectable markers also include resistance to antibioticssuch as kanamycin or tetracycline.

Other nucleic acid sequences encoding additional functions may also bepresent in the vector, as is known in the art. For instance, whenAgrobacterium is the host, T-DNA sequences may be included to facilitatethe subsequent transfer to and incorporation into plant chromosomes.

Plants for Genetic Engineering

The present invention comprehends the genetic manipulation of aNicotiana plant for regulating nicotinic alkaloid synthesis viaintroducing a polynucleotide sequence that encodes an enzyme in thepathway for nicotinic alkaloid synthesis. Accordingly, the presentinvention provides methodology and constructs for reducing or increasingnicotinic alkaloid synthesis. Additionally, the invention providesmethods for producing nicotinic alkaloids and related compounds innon-nicotine producing plants, such as Arabidopsis thaliana.

“Genetically engineered” (GE) encompasses any methodology forintroducing a nucleic acid or specific mutation into a host organism Forexample, a tobacco plant is genetically engineered when it istransformed with a polynucleotide sequence that increases expression ofa gene, such as MPO1 or MPO2, and thereby increases nicotine levels.Likewise, a plant is genetically engineered when it is transformed witha polynucleotide sequence that reduces expression of a gene, such asMPO1 or MPO2. In contrast, a tobacco plant that is not transformed witha polynucleotide sequence is a control plant and is referred to as a“nontransformed” plant.

In the present context, the “genetically engineered” category includes“transgenic” plants and cells (see definition, infra), as well as plantsand cells produced by means of targeted mutagenesis effected, forexample, through the use of chimeric RNA/DNA oligonucleotides, asdescribed by Beetham et al., Proc. Nat'l. Acad. Sci. USA 96: 8774-8778(1999) and Zhu et al., loc. cit. at 8768-8773, or so-called“recombinagenic olionucleobases,” as described in PCT application WO03/013226. Likewise, a genetically engineered plant or cell may beproduced by the introduction of a modified virus, which, in turn, causesa genetic modification in the host, with results similar to thoseproduced in a transgenic plant, as described herein. See, e.g., U.S.Pat. No. 4,407,956. Additionally, a genetically engineered plant or cellmay be the product of any native approach (i.e., involving no foreignnucleotide sequences), implemented by introducing only nucleic acidsequences derived from the host species or from a sexually compatiblespecies. See, e.g., U.S. published application No. 2004/0107455.

“Plant” is a term that encompasses whole plants, plant organs (e.g.leaves, stems, roots, etc.), seeds, differentiated or undifferentiatedplant cells, and progeny of the same. Plant material includes, withoutlimitation, seeds suspension cultures, embryos, meristernatic regions,callus tissues, leaves, roots, shoots, stems, fruit, gametophytes,sporophytes, pollen, and microspores. The class of plants which can beused in the present invention is generally as broad as the class ofhigher plants amenable to genetic engineering techniques, including bothmonocotyledonous and dicotyledonous plants, as well as gymnosperms. Apreferred nicotine producing plant includes Nicotiana, Duboisia,Solanum, Anthocercis, and Salpiglessis genera in the Solanaceae or theEclipta and Zinnia genera in the Compositae.

“Tobacco” refers to any plant in the Nicotiana genus that producesnicotinic alkaloids. Tobacco also refers to products comprising materialproduced by a Nicotiana plant, and therefore includes, for example,expanded tobacco, reconstituted tobacco, cigarettes, cigars, chewingtobacco or forms of smokeless tobacco, snuff and snus made from GEincreased-nicotine tobacco. Examples of Nicotiana species include butare not limited to the following: Nicotiana acaulis, Nicotianaacuminata, Nicotiana acuminata var. multzjlora, Nicotiana africana,Nicotiana alata, Nicotiana amplexicaulis, Nicotiana arentsii, Nicotianaattenuata, Nicotiana benavidesii, Nicotiana benthamiana, Nicotianabigelovii, Nicotiana bonariensis, Nicotiana cavicola, Nicotianaclevelandii, Nicotiana cordifolia, Nicotiana corymbosa, Nicotianadebneyi, Nicotiana excelsior, Nicotiana forgetiana, Nicotiana fragrans,Nicotiana glauca, Nicotiana glutinosa, Nicotiana goodspeedii, Nicotianagossei, Nicotiana hybrida, Nicotiana ingulba, Nicotiana kawakamii,Nicotiana knightiana, Nicotiana langsdorffiii, Nicotiana linearis,Nicotiana longiflora, Nicotiana maritima, Nicotiana megalosiphon,Nicotiana miersii, Nicotiana noctiflora, Nicotiana nudicaulis, Nicotianaobtusifolia, Nicotiana occidentalis, Nicotiana occidentalis subsp.hesperis, Nicotiana otophora, Nicotiana paniculata, Nicotianapauczjlora, Nicotiana petunioides, Nicotiana plumbaginifolia, Nicotianaquadrivalvis, Nicotiana raimondii, Nicotiana repanda, Nicotianarosulata, Nicotiana rosulata subsp. ingulba, Nicotiana rotundifolia,Nicotiana rustica, Nicotiana setchellii, Nicotiana simulans, Nicotianasolanifolia, Nicotiana spegauinii, Nicotiana stocktonii, Nicotianasuaveolens, Nicotiana sylvestris, Nicotiana tabacum, Nicotianathyrsiflora, Nicotiana tomentosa, Nicotiana tomentosifomis, Nicotianatrigonophylla, Nicotiana umbratica, Nicotiana undulata, Nicotianavelutina, Nicotiana wigandioides, and Nicotiana x sanderae.

The Erythroxylaceae (or coca family) is a family of flowering plantsconsisting of 4 genera and about 240 species. The best-known species byfar is the coca (Erythroxylum coca). It has been previously reportedthat when labeled 4-methylaminobutanal diethyl acetal (an acetalderivative of N-methylpyrrolinium cation) was fed to the leaf ofErythroxylum coca, the label was incorporated into the tropane moiety ofcocaine. Leete, Planta Med. 56: 339-352 (1990). Therefore, it isreasonable to presume that MPO genes of the present invention areinvolved in the formation of cocaine.

In the present description, “tobacco hairy roots” refers to tobaccoroots that have T-DNA from an Ri plasmid of Agrobacterium rhizogenesintegrated in the genome and grow in culture without supplementation ofauxin and other phytohormones. Tobacco hairy roots produce nicotinicalkaloids as roots of a tobacco plant do. These types of roots arecharacterized by fast growth, frequent branching, plagiotropism, and theability to synthesize the same compounds as the roots of the intactplant. David et al., Biotechnology 2: 73-76. (1984). Roots of Solanaceaeplants are the main site of tropane alkaloid biosynthesis, and hencehairy root cultures also are capable of accumulating high levels ofthese metabolites. For example, see Oksman-Caldentey & Arroo,“Regulation of tropane alkaloid metabolism in plants and plant cellcultures,” in METABOLIC ENGINEERING OF PLANT SECONDARY METABOLISM pp.253-81 (Kluwer Academic Publishers, 2000).

Non-Nicotine Producing Cells for Genetic Engineering

The invention contemplates genetically engineering “non-nicotineproducing cells” with a nucleic acid sequence encoding an enzymeinvolved in the production of nicotinic alkaloids. Non-nicotineproducing cells refer to a cell from any organism that does not producenicotine. Illustrative cells include but are not limited to plant cells,such as Atropa belladonna, Arabidopsis thalianu, as well as insect,mammalian, yeast, fungal, algal, or bacterial cells. Suitable host cellsare discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS INENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).

“Insect cell” refers to any insect cell that can be transformed with agene encoding a nicotine biosynthesis enzyme and is capable ofexpressing in recoverable amounts the enzyme or its products.Illustrative insect cells include Sf9 cells (ATCC CRL 171 1).

“Fungal cell” refers to any fungal cell that can be transformed with agene encoding a nicotine biosynthesis enzyme and is capable ofexpressing in recoverable amounts the enzyme or its products.Illustrative fungal cells include yeast cells such as Saccharomycescerivisae (Baldari, et al., 1987. EMBO J. 6: 229-234) and Pichiapastoris (e.g. P. pastoris KM714 available from Invitrogen). Cells offilamentous fungi such as Aspergillus and Trichodemza may also be used.Archer, et al., Antonie van Leeuwenhoek 65: 245-250 (2004).

“Bacterial cell” refers to any bacterial cell that can be transformedwith a gene encoding a nicotinic alkaloid biosynthesis enzyme and iscapable of expressing in recoverable amounts the enzyme or its products.Illustrative bacterial cells include E. coli, such as E. coli strainM15/rep4, which is available commercially from QIAGEN.

“Mammalian cell” refers to any mammalian cell that can be transformedwith a gene encoding a nicotine biosynthesis enzyme and is capable ofexpressing in recoverable amounts the enzyme or its products.Illustrative mammalian cells include Chinese hamster ovary cells (CHO)or COS cells. Mammalian cells may also include a fertilized oocyte or anembryonic stem cell into which nicotinic alkaloid biosynthesisenzyme-coding sequences have been introduced. Such host cells can thenbe used to create non-human transgenic animals. Examples of systems forregulated expression of proteins in mammalian cells include Clontech'sTet-Off and Tet-On gene expression systems and similar systems. Gossenand Bujard, Proc. Natl. Acad. Sci. USA 89: 55475551 (1992).

“Algae cell” refers to any algae species that can be transformed with agene encoding a nicotine biosynthesis enzyme without adversely affectingnormal algae development or physiology. Illustrative algae cells includeChlamydomonas reinhardtii (Mayfield and Franklin, Vaccine 23: 1828-1 832(2005).

Because production of nicotinic alkaloids in an insect cell couldadversely affect insect growth and development, an inducible expressionsystem may mitigate adverse affects. For example, insect cells may befirst grown under non-inducing conditions to a desired state and thenexpression of the enzyme is induced.

Additionally, cells expressing nicotinic alkaloid biosynthesis genes maybe supplied with precursors to increase substrate availability fornicotinic alkaloid synthesis. Cells may be supplied with analogs ofprecursors which may be incorporated into analogs of naturally occurringnicotinic alkaloids.

Transformation and Selection

While nicotine is the major alkaloid in N. tabacum and some otherspecies in the Nicotiana genus, other plants have nicotine-producingability, including, for example, Duboisia, Solanum, Anthocercis andSalpiglessis genera in the Solanaceae, and Eclipta and Zinnia genera inthe Compositae. Using the inventive constructs and methods, nicotine maybe produced in non-nicotine producing plants, such as Atropa belladonnaand Arabidopsis thaliana, and cells, such as insect, fungal, andbacterial cells.

For the purposes of this description, a plant or non-nicotine producingcell, such as a fungal cell, may be transformed with a plasmidcomprising one or more sequences, each operably linked to a promoter.For example, an illustrative vector may comprise a MPO1 sequenceoperably linked to a promoter. Likewise, the plasmid may comprise a MPO1sequence operably linked to a promoter and an MPO2 sequence operablylinked to a promoter. Alternatively, a plant or non-nicotine producingcell may be transformed with more than one plasmid. For example, a plantor non-nicotine producing cell may be transformed with a first plasmidcomprising a QPT sequence operably linked to a promoter, which isdistinct from a second plasmid comprising an MPO1 or MPO2 sequence. Thefirst and second plasmids or portions thereof may be introduced into thesame cell.

Plant Transformation

“Transgenic plant” refers to a plant that comprises a nucleic acidsequence that also is present per se in another organism or species orthat is optimized, relative to host codon usage, from another organismor species. Both monocotyledonous and dicotyledonous angiosperm orgymnosperm plant cells may be transformed in various ways known to theart. For example, see Klein et al., Biotechnology 4: 583-590 (1993);Bechtold et al., C. R. Acad. Sci. Paris 3 16: 1 194-1 199 (1993); Bentet al., Mol. Gen. Genet. 204: 383-396 (1986); Paszowski et al., EMBO J.3: 2717-2722 (1984); Sagi et al., Plant Cell Rep. 13: 262-266 (1994).Agrobacterium species such as A. tumefaciens and A. rhizogenes can beused, for example, in accordance with Nagel et al., Microbial Lett 67:325 (1990). Additionally, plants may be transformed by Rhizobium,Sinorhizobium or Mesorhizobium transformation. Broothaerts et al.,Nature 433:629-633 (2005).

For example, Agrobacterium may be transformed with a plant expressionvector via, e.g., electroporation, after which the Agrobacterium isintroduced to plant cells via, e.g., the well known leaf-disk method.Additional methods for accomplishing this include, but are not limitedto, electroporation, particle gun bombardment, calcium phosphateprecipitation, and polyethylene glycol fusion, transfer into germinatingpollen grains, direct transformation (Lorz et al., Mol. Genet. 199:179-182 (1985)), and other methods known to the art. If a selectionmarker, such as kanamycin resistance, is employed, it makes it easier todetermine which cells have been successfully transformed. Marker genesmay be included within pairs of recombination sites recognized byspecific recombinases such as cre or flp to facilitate removal of themarker after selection. See U.S. published application No. 2004/0143874.

Transgenic plants without marker genes may be produced using a secondplasmid comprising a nucleic acid encoding the marker, distinct from afirst plasmid that comprises an MPO1 or MPO2 sequence. The first andsecond plasmids or portions thereof are introduced into the same plantcell, such that the selectable marker gene that is transientlyexpressed, transformed plant cells are identified, and transformedplants are obtained in which the MPO1 or MPO2 sequence is stablyintegrated into the genome and the selectable marker gene is not stablyintegrated. See U.S. published application No. 2003/0221213. The firstplasmid that comprises a MPO1 or MPO2 sequence may optionally be abinary vector with a T-DNA region that is completely made up of nucleicacid sequences present in wild-type non-transgenic N. tabacum orsexually compatible Nicotiana species.

The Agrobacterium transformation methods discussed above are known to beuseful for transforming dicots. Additionally, de la Pena et al., Nature325: 274-276 (1987), Rhodes et al., Science 240: 204-207 (1988), andShimamato et al., Nature 328: 274-276 (1989) have transformed cerealmonocots using Agrobacterium. Also see Bechtold et al., C. R. Acad. Sci.Paris 3 16 (1994), illustrating vacuum infiltration forAgrobacterium-mediated transformation.

Methods of regenerating a transgenic plant from a transformed cell orculture vary according to the plant species but are based on knownmethodology. For Genetically engineered plants are selected that haveincreased expression of at least one of MPO1 and MPO2. Additionally, theinventive genetically engineered plants may have increased nicotinelevels and yield. The inventive genetically engineered plants mayoptionally have genetically engineered increased expression of one ormore of A622, NBB1, PMT, and QPT in addition to increased expression ofat least one of MPO1 and MPO2.

Non-Nicotine Producing Cell Transformation

Constructs according to the invention may be used to transform any cell,using a suitable transformation technique, such asAgrobacterium-mediated transformation for plant cells, particlebombardment, electroporation, and polyethylene glycol fusion, calciumphosphate transfection, DEAE-dextran mediated transfection, or cationiclipid-mediated transfection.

Non-nicotine producing cells may be transformed with nucleic acidconstructs of the present invention without the use of a selectable orvisible marker and transgenic organisms may be identified by detectingthe presence of the introduced construct. The presence of a protein,polypeptide, or nucleic acid molecule in a particular cell can bemeasured to determine if, for example, a cell has been successfullytransformed or transfected. For example, and as routine in the art, thepresence of the introduced construct can be detected by PCR or othersuitable methods for detecting a specific nucleic acid or polypeptidesequence. Additionally, transformed cells may be identified byrecognizing differences in the growth rate or a morphological feature ofa transformed cell compared to the growth rate or a morphologicalfeature of a non-transformed cell that is cultured under similarconditions. See WO 2004/076625.

For the purposes of the present description non-nicotine geneticallyengineered cells are selected that express at least one of MPO1 and MPO2heterologously.

Quantifying Akaloid Content

A. Reduced Alkaloids

Pursuant to one aspect of the invention, genetically engineered plantsand cells are characterized by reduced alkaloid content.

A quantitative reduction in alkaloid levels can be assayed by severalmethods, as for example by quantification based on gas-liquidchromatography, high performance liquid chromatography,radio-immunoassays, and enzyme-linked immunosorbent assays. In thepresent invention, nicotinic alkaloid levels were measured by gas-liquidchromatography equipped with a capillary column and an FID detector, asdescribed in Hibi et al., Plant Physiology 100: 826-35 (1992).

In describing a plant of the invention, the phrase “reduced nicotine” or“reduced nicotinic alkaloid content” refers to a decrease in the amountof nicotinic alkaloid in the plant or cell when compared with anon-transformed control plant or cell. A reduced-nicotine plantencompasses a genetically engineered plant that contains less than half,preferably less than 25%, and more preferably less than 20% or less than10% of the nicotine content of a control plant of the same variety. Areduced-nicotine plant also includes genetically engineered plants, suchas Nicotiana, Duboisia, Solanum, Anthocercis and Salpiglessis genera inthe Solanaceae or the Eclipta and Zinnia genera in the Compositae, thatcontain less total alkaloids compared with a control plant.

A reduced-alkaloid plant encompasses a genetically engineered plant thatcontains less than half, preferably less than 25%, and more preferablyless than 20% or less than 10% of the “total alkaloid content” of acontrol plant of the same variety.

A reduced-anabasine plant encompasses a genetically engineered plantthat contains less than half, preferably less than 25%, and morepreferably less than 20% or less than 10% of the anabasine content of acontrol plant of the same variety.

A reduced-anatalline plant encompasses a genetically engineered plantthat contains less than half, preferably less than 25%, and morepreferably less than 20% or less than 10% of the anatalline content of anon-transgenic control plant of the same variety.

B. Increased Alkaloids

In one aspect of the invention, genetically engineered plants and cellsare characterized by increased alkaloid content. Transformed nicotineproducing cells are characterized by increased nicotinic alkaloidproduction.

In describing a plant of the invention, the phrase “increased alkaloidcontent” or “increased nicotinic alkaloid content” refers to an increasein the amount of alkaloid or nicotinic alkaloids in the plant or cellwhen compared with a non-transformed control plant or cell. “Increasednicotine plant” encompasses a genetically engineered plant that has anincrease in nicotine content greater than 10%, and preferably greaterthan 50%, 100%, or 200% of the nicotine content of a control plant ofthe same species or variety. “Increased alkaloid plant” encompasses agenetically engineered plant that has an increase in alkaloid contentgreater than 10%, and preferably greater than 50%, 100%, or 200% of thealkaloid content of a control plant of the same species or variety.Plants of the Solonaceae and Erythroxylaceae are examples of plants thatcan be genetically engineered with MPO1 or MPO2 to increase alkaloids.

A quantitative increase in nicotinic alkaloid levels can be assayed byseveral methods, as for example by quantification based on gas-liquidchromatography, high performance liquid chromatography,radio-immunoassays, and enzyme-linked immunosorbent assays. In thepresent invention, nicotinic alkaloid levels were measured by gas-liquidchromatography equipped with a capillary column and an FID detector, asdescribed in Hibi et al., Plant Physiology 100: 826-35 (1992).

Quantifying Yield

In one aspect, the genetically engineered plants and cells of theinvention are characterized by increased nicotinic alkaloid content andyield. Increased nicotinic alkaloid production in the geneticallyengineered plants is preferably achieved by overexpressing one or morenicotine biosynthesis pathway genes, including at least one of MPO1 andMPO2.

In describing a plant of the invention, the phrase “increased yield” or“high yielding” refers to an increase in the amount of yield of a plantor crop of said plant when compared to an increased-nicotine controlplant or crop of said plant. “Increased yield plant” encompasses agenetically engineered plant that yields the same as a non-increasednicotine plant, or yields a greater amount than an increased-nicotineplant, preferably greater than 110%, and more preferably greater than125% of the yield of an increasesd-nicotine plant of the same species orvariety.

A quantitative increase in photosynthetic efficiency can be assayed byseveral methods, as for example by quantifying photosynthetic rates,such as gas exchange and CO₂ fixation, and chlorophyll florescence.Miyagawa et al., Plant Cell Physiol. 41, 31 1-320 (2000). Photosyntheticrates may also be quantified by measuring metabolite and carbohydratelevels as described by Leegood, Carbon Metabolism In Photosynthesis andproduction in a changing environment: a field and laboratory manual(eds. Hall, Scurlock, Bolhar-Nordenkampf, Leegood, & Long) 247-267(Chapman & Hall, London; 1993). Alternatively, photosynthetic activitymay be calculated based on enzyme activity, such as Rubisco activity.Portis, A. R. J. Exp. Bot. 46: 1285-1291 (1995).

Of course, increased yield can be determined by measuring more readilydiscernible characteristics, including but not limited to plant height,weight, leaf size, number of seeds produced, and seed weight.

Reduced-Nicotine Products

The present invention provides a transgenic plant or cell havingreducednicotinic-alkaloid levels. For example, the instant inventioncontemplates reducing nicotine levels by suppressing at least one ofMPO1 and MPO2 expression. Following selection of a transgenic planthaving suppression of at least MPO1 or MPO2 and reduced-nicotinecontent, a variety of products may be made from such a plant.

Because the present invention provides a method for reducing alkaloids,TSNAs may also be reduced because there is a significant, positivecorrelation between alkaloid content in tobacco and TSNA accumulation.For example, a significant correlation coefficient between anatabine andNAT was 0.76. Djordjevic et al., J. Agric. Food Chem., 37: 752-756(1989). TSNAs are a class of carcinogens that are predominantly formedin tobacco during curing, processing, and smoking. Hoffman et al., J.Natl. Cancer Inst. 58, 1841-4 (1977); Wiernik et al., Recent Adv. Tob.Sci, 21: 39-80 (1995). Nitrosamines, containing the organic functionalgroup, N—N═O, are formed from the facile addition of an N═O group by anitrosating agent to a nitrogen of a secondary or tertiary amine. Thisparticular class of carcinogens is found only in tobacco although theycould potentially occur in other nicotinic alkaloid-containing products.

TSNAs are considered to be among the most prominent carcinogens incigarette smoke and their carcinogenic properties are well documented.See Hecht, S. Mutat. Res. 424:127-42 (1999); Hecht, S. Toxicol. 11,559-603 (1998); Hecht, S., et al., Cancer Surv. 8, 273-294 (1989). TSNAshave been cited as causes of oral cancer, esophageal cancer, pancreaticcancer, and lung cancer (Hecht & Hoffman, IARC Sci. Publ. (105) 54-61(1991)). In particular, TSNAs have been implicated as the causativeagent in the dramatic rise of adenocarcinorna associated with cigarettesmoking and lung cancer (Hoffmann et al., Crit. Rev. Toxicol. 26, 199-211 (1996)).

The four TSNAs considered to be the most important by levels of exposureand carcinogenic potency and reported to be possibly carcinogenic tohumans are N′-nitrosonornicotine (NNN),4-methylnitrosoamino-1-(3-pyridyl)-1-butanone (NNK), N′-nitrosoanatabine(NAT) and N′-nitrosoanabasine (NAB) Reviewed in IARC Monographs of theEvaluation of Carcinogenic Risk to Humans. Lyon (France) Vol 37, pp.205-208 (1985). These TSNAs are formed by N-nitrosation of nicotine andof the minor Nicotiana alkaloids that include nornicotine, anatabine,and anabasine.

The following levels of alkaloid compounds have been reported formainstream smoke of non-filter cigarettes (measured in yg/cigarette):nicotine: 100-3000, nornicotine: 5-150, anatabine: 5-15, Anabasine: 5-12(Hoffmann et al., Chem. Res. Toxicol. 14:7:767-790 (2000)). Mainstreamsmoke of U.S. cigarettes, with or without filter tips, contain (measuredin ng/cigarette): 9-180 ng NNK, 50-500 ng NNN, 3-25 ng NAB and 55-300 ngNAT. Hoffmann, et al., J. Toxicol. Environ. Health 41: 1-52 (1994). Itis important to note that the levels of these TSNAs in sidestream smokeare 5-10 fold above those in mainstream smoke. Hoffmann, et al (1994).

Xie et al. (Recent Advances in Tobacco Science 30: 17-37 (2004))reported that Vector 21-41, a genetically engineered reduced-nicotinetobacco with down-regulation of QPT, has a total alkaloid level of about2300 ppm, which is less than 10 percent of the wild-type tobacco. SeeU.S. Pat. No. 6,907,887. Mainstream smoke from cigarettes made from theVector 21-41 tobacco had less than 10 percent of NNN, NAT, NAB, and NNKcompared to the levels of a standard full flavor cigarette produced fromwild-type tobacco.

Strategies for reducing TSNAs by reducing the corresponding tobaccoalkaloid precursors is currently a main focus of agricultural tobaccoresearch. Sirninszky et al., Proc. Natl. Acad. Sci. USA 102(41)14919-14924 (2005). To reduce formation of all TSNAs there is an urgentneed to reduce the precursor nicotinic alkaloids as much as possible bygenetic engineering.

A reduced-nicotine tobacco product may be in the form of leaf tobacco,shredded tobacco, cut tobacco and tobacco fractions. A reduced-nicotinetobacco product may include cigarette tobacco, cigar tobacco, snuff,chewing tobacco, pipe tobacco, and cigarettes made from geneticallyengineered reduced-nicotine tobacco for use in smoking cessation.Reduced-nicotine tobacco may also be used to produce reconstitutedtobacco (Recon). Recon is produced from tobacco stems and/or smallerleaf particles by a process that closely resembles paper making. Thisprocess entails processing the various tobacco portions that are to bemade into Recon and cutting the tobacco into a size and shape thatresembles cut rag tobacco made from whole leaf tobacco. This cut reconthen is mixed with cut-rag tobacco and is ready for cigarette making.

In addition to traditional tobacco products, such as cigarette and cigartobacco, reduced-nicotine tobacco can be used as source for protein,fiber, ethanol, and animal feeds. See WO/2002/098208. For example,reduced-nicotine tobacco may be used as a source of Rubisco (ribulosebisphosphate carboxylase-oxygenase, or fraction 1 protein) becauseunlike other plants, tobacco-derived Rubisco can be readily extracted incrystalline form. With the exception of slightly lower levels ofmethionine, Rubisco's content of essential amino acids equals or exceedsthat of the FAO Provisional Pattern. Ershoff, B. H., et al. Society forExperimental Biology and Medicine 157:626-630 (1978); Wildman, S. G.Photosynthesis Research 73:243-250 (2002).

For biofuels to replace a sizable portion of the world's dependence onnonrenewable energy sources, co-products, such as Rubisco, are requiredto help defray the cost of producing this renewable energy. Greene etal., Growing Energy. How Biofuels Can End America's Oil Dependence;National Resources Defense Counsel (2004). Thus, the greater reductionin nicotinic alkaloids in tobacco, the greater the likelihood of asuccessful tobacco biomass system.

Increased-Nicotine Products

The present invention provides a genetically engineered plant havingincreased-nicotine levels, as well as a genetically engineerednon-nicotine producing cell that produces nicotine or related compounds,where said cell is derived from an organism that does not producenicotine. A variety of products may be made from such a geneticallyengineered plant. Likewise, products can be made from cells that aregenetically engineered for production of nicotine or related compounds.

Herbivore-Resistant Plant

Nicotine serves as a natural pesticide which helps protect tobaccoplants from damage by pests. It has been show that conventionally bredor transgenic low-nicotine tobacco have increased susceptibility toinsect damage. Legg, P. D., et al., Can. J. Cyto., 13:287-291 (197 1);Voelckel, C., et al., Chemoecology 11: 121-126 (2001); Steppuhn, A., etal., PLoS Biol, 2(8): e217: 1074-1080 (2004). Using the inventivemethods and constructs, increased-nicotine plants may be produced thathave increased resistance to insect and other pest damage.

Increased-Nicotine Tobacco Products

The inventive constructs and methods may be used to produce, forexample, cigarettes, cigars, and other traditional tobacco products suchas snuff and snus. Additionally, increased-nicotine cigarettes may beproduced that have reduced-exposure to smoke components, such as tar,yet have similar or increased nicotine deliveries as conventionalcigarettes.

In the present description, an increased-nicotine tobacco product may bein the form of leaf tobacco, shredded tobacco, cut rag tobacco, groundtobacco, reconstituted tobacco, expanded or puffed tobacco and tobaccofractions including, for example, nicotine. An increased-nicotinetobacco product may include cigarettes, cigars, pipe tobaccos, and anyform of smokeless tobacco such as snuff, snus, or chewing tobacco.

Blending different tobacco types or cultivars within a tobacco productsuch as a cigarette is common in tobacco art. It will therefore beappreciated that increased-nicotine tobacco could be blended at anypercentage with non-transformed tobacco to obtain any level of desirednicotine content, up to the nicotine content of the increased nicotinetobacco utilized, to manufacture a tobacco product.

Increased nicotine cigarettes are particularly advantageous becausestudies demonstrate that when nicotine is increased, smokers inhale lesstar and carbon monoxide. See Armitage et al., Psychopharmacology96:447-453 (1988); Fagerström, Psychopharmacology 77: 164-167 (1982);Russell, Nicotine and Public Health 15:265-284 (2000) and Woodman etal., European Journal of Respiratory Disease 70:3 16-321 (1987).

Cigarette smoke is an extremely complex mixture of more than 4,000different compounds. Green & Rodgman, Recent Advances in Tobacco Science22: 131-304 (1996); 10M Report, page 9 of executive summary. Cigarettesmoke is made up of two phases: a particulate phase, which is commonlycalled “tar” or total particulate matter, and a vapor phase, whichcontains gases and semi-volatile compounds. A common definition for“tar” is “nicotine-free dry smoke” or “nicotine-free dry particulatematter” (NFDPM) captured by a Cambridge pad when a cigarette is machinesmoked. More specifically, “tar” is the total particulate matterisolated from smoke, excluding water and nicotine. Tar makes up lessthan ten percent of the weight of cigarette smoke. The tar componentcontains the majority of the most harmful smoke compounds.

Analytical methods combined with sensitive biological assays have led tothe identification of 69 carcinogens in tobacco smoke. See THE CHANGINGCIGARETTE: CHEMICAL STUDIES AND BIOASSAYS Chapter 5, Smoking and TobaccoControl Monograph No. 13 (NIH Pub. No. 02-5074, October 2001). It hasbecome clear to researchers, however, that not all components ofcigarette smoke have equal toxicity. Notably, the first U.S. SurgeonGeneral's report on smoking in 1964 came to the conclusion that nicotinewas probably not toxic at the levels inhaled by smokers, with theimplication that the source of the primary pharmacologic reward tosmokers was not of immediate concern. The Surgeon General's 1964 reportstated, at page 74, that “[t]here is no acceptable evidence thatprolonged exposure to nicotine creates either dangerous functionalchanges of an objective nature or degenerative diseases.”

In fact, the U.S. Food and Drug Administration allows the sale ofnicotine replacement products such as patches and chewing gum for use insmoking cessation therapy. These products may deliver more nicotine inone day than a pack of cigarettes. Page 167 of the 10M Report states,“Many studies of nicotine suggest that nicotine is unlikely to be acancer-causing agent in humans or, at worst, that its carcinogenicitywould be trivial compared to that of other components of tobacco. Theconsideration of nicotine as a carcinogenic agent, if at all, is trivialcompared to the risk of other tobacco constituents.”

Cigarettes are generally rated by the FTC (in the U.S.) or ISO smokingmachine methods which determine, for example, the amount of tar andnicotine generated when a cigarette is smoked by a machine understandardized conditions. See Pillsbury et al., J Assoc. Off. AnalyticalChem. (1969); ISO: 4387 (1991). Most commercial cigarettes generallyyield about 10 to 15 parts “tar” to every 1 part nicotine, measured inmilligrams, as analyzed in PCT application WO 2005/018307. Many publichealth officials believe that the current FTC/ISO machine smoking regimeis flawed since these methodologies fail to take into account humansmoking behavior which is primarily driven by nicotine seeking. In otherwords, these methods don't consider compensatory smoking. Compensatorysmoking or compensation, as it is also called, essentially means oversmoking (smoking more intensively) due to the reduced presence ofnicotine in tobacco smoke or under smoking (smoking less intensively)due to the increased presence of nicotine. See Benowitz, N. CompensatorySmoking of Low Yield Cigarettes, In Risks Associated with SmokingCigarettes with Low Machine-Measured Yields of Tar and Nicotine NCISmoking and Tobacco Control Monograph 13 (2001).

Novel smoking-machine methods are currently being evaluated, especiallythose that consider compensatory smoking of low-yield brands. An exampleis a method involving the adjustment of smoking parameters so thatbrands with lower ISO nicotine yields are machine smoked more intensely.Kozlowski and O'Connor Lancet 355: 2159 (2000). Other proposed methodsmeasure yields of toxins on a per nicotine unit basis or at a defined“target” nicotine yield. This is achieved, for example, bysystematically varying puff volume, puff interval, and blockage ofventilation holes until the target nicotine yield is reached. Cigarettescan then be rated on the effort required to get the target nicotineyield as well as on toxin delivery at that yield. Consumers wouldbenefit from these smoking-machine methods since comparisons of specifictoxins among different brands could be evaluated.

Studies have suggested that many smokers Inhale just as much smoke withmost “light” and “ultra-light” cigarettes as full flavor cigarettes(Gori and Lynch, Regulatory Toxicology and Pharmacology 5:3 14-326).Smokers may compensate or smoke lower-yield cigarettes (per the FTC orISO method) more aggressively (than higher-yield cigarettes) in order toobtain their desired nicotine impact and mouth feel of smoke, which areimportant sensory properties. Rose, J. E. “The role of upper airwaystimulation in smoking,” in Nicotine Replacement: A Critical Evaluation,pp. 95-106, 1988.

The manner in which a smoker may compensate include the frequency ofpuffs per cigarette and volume of smoke inhalation of such puffs,duration of the smoke inhalation being held before exhaling, number ofcigarettes smoked within a specified time period, and the percentage ofeach cigarette that is smoked (how far down the cigarette is smoked).

When the percentage of nicotine per unit of inhaled smoke volumeincreases, many smokers may compensate and inhale less smoke. Gori G.B., Virtually Safe Cigarettes. Reviving an opportunity once tragicallyrejected. IOS Press. Amsterdam, (2000). The higher the percentage ofnicotine in cigarette tobacco, the higher the percentage of nicotine incigarette smoke. More specifically, the higher the percentage ofnicotine in a cigarette's filler, the higher the percentage of nicotinein cigarette smoke. “Filler” means any smokable material that is rolledwithin a cigarette or cigarette-like device and includes (a) alltobaccos, including but not limited to reconstituted and expandedtobaccos, (b) any non-tobacco substitutes that may accompany (a); (c)tobacco casings, (d) other additives including flavorings that (a), (b)or (c) are supplemented with. A cigarette-like device is any devicespecifically intended to deliver nicotine through an alternative “smoke”aerosol formed by heating tobacco materials. Characteristics of suchdevices are that they contain a high content of glycerin or a glycerinsubstitute with minimal or no combustion pyrolysis. Glycerin whenheated, vaporizes rapidly and forms an aerosol that can be inhaled andis very similar in appearance and feel to conventional cigarette smoke.

Therefore, the nicotine content of tobacco filler contained within acigarette or cigarette-like device, all other factors held constant(including but not limited to, the type of filter, cigarette paperincluding its porosity, plug wraps, and tipping paper utilized, and theamount of filter ventilation), would roughly have to double for acorresponding two-fold increase of nicotine in mainstream cigarettesmoke. Further, the nicotine content of tobacco filler contained withina cigarette or cigarette-like device, all other factors held constant,would roughly have to triple for a corresponding three-fold increase ofnicotine in mainstream cigarette smoke. The calculations in this sectionrefer to protonated nicotine in the mainstream smoke of a cigaretteaugmented with the described increase in nicotine levels and not “free”or “volatile” nicotine.

In one preferred embodiment of the invention, a reduced-exposurecigarette is manufactured that comprises an increased-nicotine tobaccoplant having up to a two-fold increase of nicotine. In another preferredembodiment of the present invention a reduced-exposure cigarette ismanufactured comprising an increased-nicotine tobacco having greaterthan a two-fold increase of nicotine.

“Curing” is the aging process that reduces moisture and brings about thedestruction of chlorophyll giving tobacco leaves a golden color and bywhich starch is converted to sugar. Cured tobacco therefore has a higherreducing sugar content and a lower starch content compared to harvestedgreen leaf. “Flue-cured tobacco” refers to a method of drying tobaccoplants in a ventilated barn with heat and is characterized by a uniquecolor, high reducing sugar content, medium to heavy in body andexceptionally smooth smoking properties. Bacon et al., Ind. Eng. Chem.44: 292 (1952).

Increased-nicotine tobacco may contain higher nitrosamines since thereis a positive correlation between alkaloid content in tobacco and TSNAaccumulation. However, U.S. Pat. No. 5,803,081, No. 6,135,121, No.6,805,134, No. 6,895,974 and No. 6,959,712 and U.S. PublishedApplications 2005/0034365, 2005/0072047, 2005/0223442, 2006/0041949, andPCT published application WO 2006/091194, and others, discussmethodology to reduce tobacco-specific nitrosamines, which can beapplied to a tobacco product that utilizes the subject invention.

Accordingly, the present invention provides constructs and methodologyfor producing cigarettes and other tobacco products containing increasednicotine levels. A desirable reduced-exposure cigarette should deliver asmoker's desired level of nicotine per cigarette as efficiently aspossible while maintaining acceptable taste. See WO 05/018307.

Reconstituted Tobacco, Expanded Tobacco and Blending

Increased-nicotine tobacco also may be used to produce reconstitutedsheet tobacco (Recon) and expanded tobacco or puffed tobacco. Recon canbe produced from the following: tobacco dust, stems, small leafparticles, other byproducts of tobacco processing and cigarettemanufacturing, and sometimes straight whole leaf. The recon process,which varies by manufacturer, closely resembles the typical paper makingprocess and entails processing the various tobacco fractions and thencutting the recon sheets into a size and shape that resembles cigarettetobacco (cut-rag tobacco).

In addition, increased-nicotine tobacco may be used, according to thepresent invention, to produce expanded tobacco, also known as puffedtobacco, which is an important component in many cigarette brands.Expanded tobacco is made through expansion of suitable gases, wherebythe tobacco is “puffed,” resulting in reduced density and greaterfilling capacity, which in turn reduces the weight of tobacco used incigarettes. By using increased-nicotine tobacco as a starting material,cigarettes made with the resultant expanded tobacco will yield reducedratios of toxic chemicals, such as tar and carbon monoxide, to nicotine.

Increased-nicotine expanded tobacco, increased-nicotine Recon, andincreased-nicotine cut-rag tobacco can be blended at any percentageamong the three or with any percentages of non-transformed expandedtobacco, non-transformed recon or non-transformed cut-rag to producecigarette filler containing varying nicotine contents. Any such blend isthen incorporated into the cigarette making process according tostandard methods known in the art.

Tobacco products other than cigarettes utilizing genetically engineeredincreased-nicotine tobacco are manufactured using any of the tobaccoplant material described herein according to standard methods known inthe art. In one embodiment, tobacco products are manufactured comprisingplant material obtained from increased-nicotine tobacco. Theincreased-nicotine content can be up to greater than three times that ofwild type cultivars.

Nicotinic Alkaloid Enzymes and Analogs

In addition to traditional tobacco products, such as cigarettes andchewing tobacco, the present invention provides methodology forproducing nicotine and nicotine analogs, as well as enzymes forsynthesis of nicotine and nicotine analogs. These compounds may beproduced by genetically engineered nicotine-producing plants andnon-nicotine producing cells, as well as in a cell-free/in vitro system.

Because recent studies suggest a role for nicotine receptors in treatinga variety of conditions and disorders, such as Alzheimer's disease,schizophrenia, central and autonomic nervous systems dysfunction, andaddictions, there is a need for nicotine receptor ligand sources. Forexample, the inventive methods and constructs may be used for producingnicotinic alkaloids. It has been shown that transgenic hairy rootcultures overexpressing PMT provide an effective means for large-scalecommercial production of scopolamine, a pharmaceutically importanttropane alkaloid. Zhang et al., Proc. Nat'l Acad. Sci. USA 101: 6786-91(2004). Accordingly, large-scale or commercial quantities of nicotinicalkaloids can be produced in tobacco hairy root culture byoverexpressing at least one of MPO1 and MPO2. Likewise, the presentinvention contemplates cell culture systems, such as bacterial or insectcell cultures, for producing large-scale or commercial quantities ofnicotinic alkaloids, nicotine analogs or nicotine precursors byexpressing at least one of MPO1 and MPO2. The cells may optionallyexpress other nicotine biosynthesis genes such as PMT, QPT, A622 andNBB1.

Additionally, products can be made directly using the activity of MPOenzymes encoded by MPO1 and MPO2. For example, recombinant MPO1 and MPO2enzymes may be used for the synthesis, or partial synthesis, ofnicotinic alkaloids or nicotinic alkaloid analogs. Accordingly,large-scale or commercial quantities of MPO1 and MPO2 can be produced bya variety of methods using the MPO1 and MPO2 genes, including extractingrecombinant enzyme from a genetically engineered plant, cell, or culturesystem, including but not limited to hairy root cultures, insect,bacterial, fungal, plant, algae, and mammalian cell culture. or invitro.

Specific examples are presented below of methods for identifyingsequences encoding MPO enzymes involved in alkaloid biosynthesis, aswell as for introducing target gene to produce plant transformants withaltered alkaloid contents. They are meant to be exemplary and not aslimitations on the present invention.

EXAMPLE 1 Identification of MPO1 and MPO2 as Genes Regulated by the nicLoci

A cDNA micro-array prepared from a Nicotiana sylvestris-derived cDNAlibrary, see Katoh et al., Proc. Japan Acad., Vol. 79, Ser. B, No. 6,pp. 151-154 (2003), was used to search for novel genes which arecontrolled by the nicotine biosynthesis regulatory NIC loci.

N. sylvestris cDNAs were amplified by PCR and spotted onto mirror-coatedslides (type 7 star, Amersham) using an Amersham Lucidea array spotter.DNA was immobilized on the slide surface by UV crosslinking (120 mJ/m2).N. tabacum Burley 21 plantlets (WT and nic1nic2) were grown onhalf-strength B5 medium supplemented with 1.5% (W/V) sucrose and 0.35%(W/V) gellan gum (Wako) in Agripot containers (Kirin).

Roots of eight-week-old plantlets were harvested, immediately frozenwith liquid nitrogen, and kept at −80° C. until use. Total RNA wasisolated from the frozen roots using a Plant RNeasy Mini kit (Qiagen),and mRNA was purified using a GenElute mRNA Miniprep kit (Sigma). cDNAwas synthesized from 0.4 pg of the purified mRNA using a Labelstar ArrayKit (Qiagen) and Cy3 or Cy5-dCTP (Amersham). cDNA hybridization to themicroarray slides and post-hybridization washes were performed using aLucida Pro hybrid-machine (Amersham). Microarrays were scanned using anFLA-8000 scanner (Fujifilm). Acquired array images were quantified forsignal intensity with ArrayGauge software (Fujifilm). cDNA probes fromwild type and nic1nic2 tobacco were labeled with Cy3 and Cy5 inreciprocal pair-wise combinations. Hybridization signals were normalizedto the total signal intensity of dyes. cDNA clones which hybridized towild-type probes more than twice as strongly as they did to nic1nic2probes were identified, and these included MPO1. Full-length MPO1 cDNAwas obtained by 5′- and 3′-RACE from total RNA of N. tabacum by using aSMART RACE cDNA Amplification Kit (Clontech). During this cloningprocedure, another cDNA clone of N. tabacum that was highly homologousto the MPO1 nucleotide sequence was found, and its full-length cDNA,obtained by 5′- and 3′-RACE as described above, was designated as MPO2.

The nucleotide sequences of the MPO1 and MPO2 cDNA inserts weredetermined on both strands using an ABI PRISM® 3100 Genetic Analyzer(Applied Biosystems) and a BigDye® Terminator v3.1 Cycle Sequencing Kit(Applied Biosystems). The nucleotide sequences of the MPO1 and the MPO2are set forth in SEQ ID NO: 1 and SEQ ID NO: 3, respectively. Therespective amino acid sequences encoded by the nucleotide sequences areset forth in SEQ ID NO: 2 and SEQ ID NO: 4. The protein sequencesinclude a putative active-site tyrosine and three histidines presumablyrequired for binding copper. The nucleotide sequences of the MPO1 ORFand the MPO2 ORF are set forth in SEQ ID NO: 5 and SEQ ID NO: 6,respectively.

EXAMPLE 2 Enzymology of MPO1

The open reading frame of the MPO1 nucleotide sequence (SEQ ID NO: 1)was amplified by PCR using the following primers:

primer 1: 5′-CGATATCAATGGCCACTACTAAACAGAAAGTGACGGCACC-3′ primer 2:5′-TCGCCGGCGTCAAAGCTTGGCCAGCAA-3′

The PCR product was first cloned into a pGEM-T Easy vector (Promega),then excised as the EcoRV-Not1 fragment, and finally cloned into theexpression vector pGEX-6P (GE Healthcare). The resultant plasmidpGEX-MPO1 was introduced into the E. coli strain Rosetta (DE3)(Novagen). The recombinant bacteria were cultured in LB mediumcontaining 50 μg/ml carbenicillin at 37° C. until the OD₆₀₀ of theculture reached 0.4, IPTG and CuSO₄ were added to the culture to thefinal concentrations of 0.1 mM and 4 mM, respectively, and the cultureswere incubated at 16° C. for 16 h. Bacteria were harvested bycentrifugation, suspended in a PBS buffer containing 10 mM DTT and 1mg/ml lysozyme, incubated at 4° C. for 2 h, and then sonicated for 3min. After centrifugation of the homogenate, the recombinant MPO1protein fused to GST was purified from the supernatant by GSTrap HP (GEHealthcare) according to the manufacturer's protocol.

To measure the amine oxidase activity, ammonia produced was measuredenzymatically using glutamate dehydrogenase (from beef liver; OrientalYeast Co., Tokyo) by monitoring the decrease in NADH at 339 nm (Kuscheand Lorenz, Methods of Enzymatic Analysis, Vol. 3, pp. 237-250 (1983)).Purified diamine oxidase from pig kidney was purchased from Sigma.Enzyme activities were calculated by data analysis software, KaleidaGraph (HULINKS). Table 1 shows that recombinant tobacco MPO1preferentially oxidatively deaminated N-methlated diamines, with thelowest K_(m) value of 0.036 mM observed for N-methylputrescine. On theother hand, pig kidney diamine oxidase preferred putrescine overN-methylputrescine. This demonstrates that MPO1 is a diamine oxidasewith a high preference for N-methylputrescine as substrate.

To determine the subunit organization of MPO1 protein, the GSTpurification tag in the fusion protein was removed by treatment withPreScission protease (GE Healthcare). The molecular weight of the nativeMPO1 protein was determined by chromatography using a calibrated TSK-GELgel filtration column (TOSOH, Tokyo) and PBS buffer. The subunit sizewas determined by measuring the molecular weight of the denatured MPO1protein by SDSPAGE. The native molecular weight of MPO1 was estimated tobe 172+/−10 kDa, whereas its denatured molecular weight was 84+/−11 kDa,which is in reasonable agreement with a molecular weight of 89 kDacalculated from the predicted amino acid sequence. This experimentindicates that MPO1 is a dimer, as are known diamine oxidases.

EXAMPLE 3 Expression Profile of MPO

MPO expression was investigated in tobacco plants by Northern blotanalysis. RNA was extracted from plant bodies which had been treatedwith methyljasmonate vapor, using Nicotiana tabacum cv. Burley 21(abbreviated below as WT) and lines in which nic1, nic2 and nic1 nic2mutations had been introduced into the Burley 21 background. Plants wereraised in a sterile sealed environment for 2 months at 25° C. with 150μmole photons/m2 of light (16 h light, 8 h dark) on ½×B5 medium (with 3%sucrose and 0.3% gellan gum). Methyl jasmonate treatment wasaccomplished by adding 0.5 mL of 100 μM methyl jasmonate to an Agripotcontainer (Kirin, Tokyo) with a solid medium volume of 80 cm³ and a gasvolume of 250 cm³ containing the plants. The root parts and leaf parts(2nd through 6th leaves from a plant with a total of 7 to 10 leaves)were collected from treated and control plants 24 h after the additionof methyl jasmonate and immediately stored frozen using liquid nitrogen.

RNA was extracted using an RNeasy Midi Kit (Qiagen) according to themanufacturer's protocol, except that polyvinyl pyrrolidone was added toa concentration of 1% to the extraction buffer. The column operation wasperformed twice to increase the purity of the RNA.

RNA blotting was carried out according to the ordinary methods given bySambrook, J. et al., Molecular Cloning, Cold Spring Harbor Laboratory,Chapter 7 (2001).

The sequence fragment from the start (1 bp) to 413 bp of the MPO1nucleotide sequence (SEQ ID NO: 1) was used as the probe template. Thetemplate was prepared by amplification from the cDNA clone using PCRusing the following primers:

primer 1: 5′-GGGTTCTCATCRCAGCTTTC primer 2: 5′-CCATCTCTGACCTCAGGTGTT

The probe was labeled with ³²P using a Bcabest labeling kit (Takara)according to the manufacturer's instructions. Hybridization wasaccomplished using ULTRAhyb (Ambion) as the buffer according to themanufacturer's protocol. Because MPO1 and MPO2 have very similarnucleotide sequences, the MPO1 probe used probably detected transcriptsof both MPO1 and MPO2. When a probe was prepared from MPO2 thehybridization patterns were similar to those obtained with the MPOIprobe (results not shown).

PMT probe was prepared from a PMT sequence cloned into a pcDNAII vectorin E. coli (Hibi et al., 1994). The plasmid was extracted and purifiedfrom the E. coli using a QIAprep Spin Miniprep Kit (Qiagen), treatedwith the restriction enzymes XbaI and Hind111 by ordinary methods, andrun through agarose gel electrophoresis, and DNA fragments having a sizeof about 1.5 kb were collected. A QIAquick Gel Extraction Kit (Qiagen)was used for collection. The collected DNA fragments were labeled with³²P by the same methods used for the MPO1 probe, and hybridized.

As FIGS. 2 and 3 clearly show, MPO and PMT have the same pattern ofexpression in tobacco plants. Both genes are regulated positively by theNIC loci and methyljasmonate, and are expressed exclusively in the rootsof tobacco plants.

Relative Expression Levels of MPO1 and MPO2

The tobacco genome contains two homologous MPO genes, MPO1 and MPO2.MPO1 contains an NsiI restriction site that is not present in MPO2, andMPO2 contains a BamHI site that is not present in MPO1. We estimated therelative levels of each transcript by semi-quantitative RT-PCR followedby specific cleavage. Using an RNeasy Kit, total RNA was isolated fromtobacco hairy roots, and first-strand cDNAs were synthesized withSuperScriptII reverse transcriptase. A pair of primers designed toanneal with sequences identical in MPO1 and MPO2 was used to amplify theintervening sequences from both cDNAs as a mixture. The PCR cycle numberwas adjusted to avoid saturated amplification. The PCR productscontaining both MPO sequences were treated with BamHI and NsiI to digestMPO2 and MPO1, respectively. The reaction products were separated on anagarose gel and stained with EtBr. Undigested MPO1 and MPO2 fragmentswere detected along with smaller digestion products. From comparing theintensities of the undigested products bands in FIG. 4 it is evidentthat expression of MPO1 was much higher than expression of MPO2 intobacco hairy roots.

EXAMPLE 4 Phylogenetic Analysis of MPO1 and MPO2

The amino acid sequences of MPO1 and MPO2 show 88% identity and 96%homology. MPO1 has 25% identity and 43% homology to the Pisum sativumamine oxidase (PSAO; Tipping A J and McPherson M J, J. Biol. Chem. Vol.270, 16939-16946 (1995)) and 27% identity and 43% homology to theArabidopsis thaliana amine oxidase (ATAO1, At4g14940; Moller S G andMcPherson M J, Plant J Vol. 13, 781-791 (1998)). A phylogenetic tree wasconstructed using the sequences of MPO1, MPO2, PSAO, ATAO1, and diamineoxidase-like proteins of Arabidopsis thaliana. The phylogenetic analysiswas performed using neighbor joining method with the CLUSTAL W program.Branch lengths reflect sequence diversity counted as the number ofsubstitutions per residue. The Arabidopsis sequences used were:Atlg31710, Atlg31690, Atlg31670, Atlg31680, At4g12280, At4g1229990,At4g14940 (ATAO1), At4g12270, Atlg62810, At3g43670, and At2g42490.

The results are shown in FIG. 5. MPO1, MPO2, and an uncharacterizedArabidopsis protein At2g42490 form a distinct clade, and are separatedfrom the other sequences, including PSAO and ATAO1, at the base of thetree. PSAO and ATAO1 both contain predicted signal peptides at theirN-termini, and are thought to be extracellular copper amine oxidases,whereas MPO1, MPO2, and At2g42490 lack predicted signal peptides.

EXAMPLE 5 MPO Suppression in Transgenic Hairy Roots

RNAi was used to down-regulate the MPO genes in tobacco hairy roots. Aportion of the MPO1 cDNA (about 400 bp in length), which is a highlyhomologous between MPO1 and MPO2, was amplified by PCR using thefollowing primers:

Primer 1: 5′ GTTAGACGCTCGAGGCGACTAACAGTG 3′ Primer 2:5′ GCATATTGTGAATTCCATAGATTGTGC 3′ Primer 3:5′ GTTAGACGGTCTAGACGACTAACAGTG 3′ Primer 4:5′ GCATATTGTGTAAGCTTTAGATTGTGC 3′

The fragment was subcloned into the pHANNIBAL vector such that twoidentical cDNA segments were placed in an inverted orientation on bothsides of a pdk intron linker to produce a transcriptional cassette thatexpresses an mRNA capable of forming a dsRNA region comprising a segmentof the MPO1 sequence. This unit was excised and inserted into the binaryvector pBI121 to obtain pBI-MPO-Ri. (see FIG. 6A)

The pBI-MPO-Ri vector was introduced into Agrobactrium rhizogenes strain15834, which was used to transform tobacco SR-1 using a leaf discmethod. Kanega et al. (1994) Plant Physiol. 105: 483-490. Afterselection on kanamycin-containing medium, transgenic hairy roots weremaintained by subculturing in liquid MS medium every two weeks. Hairyroots that were growing well were used for further analyses.

To confirm the suppression of the MPO genes, we carried out RT-PCRanalysis of expression. Root tissue was frozen in liquid nitrogen andtotal RNA was isolated using an RNeasy Kit (Qiagen). First-strand cDNAswere reverse-transcribed using SuperScriptII (Invitrogen). PCRamplifications were done with following sets of primers:

for both MPO1 and MPO2:

5′-CCTTTGGACCCTTTATCTGCTGC-3′ and 5′-GGTCTTGCATATCCATTTTCCATTGG-3′for MPO1 alone:

5′-CAGCTTTCTTCCTAGCTAAGC-3′ and 5′-CTCTCTGTCCTGAATACAAGTGG-3′for MPO2 alone:

5′-TGGAAGTTTGCCTGTTGTGTGTC-3′ and 5′-AAGCTGGTACCTGGTCCAAACTG-3′

In three lines (R10, R11, and R39) out of 11 independent hairy rootlines examined, the transcript levels of MPO genes were significantlydecreased. See FIG. 7A. In these lines the transcript levels of bothMPO1 and MPO2 were decreased, but the levels of other nicotinebiosynthetic genes (PMT and QPT). See FIG. 7B. Consistent with thereduced MPO RNA level, the MPO enzyme activities were significantlylower in these lines than in the control lines. See FIG. 8.

We analyzed the alkaloid contents in the MPO down-regulated lines.Alkaloids were extracted from hairy roots 7 days after subculture andquantified by GLC as described in the previous section. In all threelines, nicotine and nornicotine were markedly decreased, while otheralkaloids (anatabine, anabasine, and anatalline) were increased. SeeFIG. 9. Anatabine was the major alkaloid accumulating in the hairy rootsin which MPO was suppressed. These results indicate that reduced MPOexpression restricts the pyrrolidine branch of tobacco alkaloidbiosynthesis, and leads to elevated levels of alkaloids that do notpossess the pyrrolidine moiety, such as anatabine, anabasine, andanatalline.

The effects of MPO suppression on the content of its substrate,N-methylputrescine, and precursors and related compounds, including thepolyamines putrescine, spermidine, and spermine, were examined.Extraction and quantification of polyamines were carried out asdescribed in Hibi et al. (1992). In brief, free and conjugated forms ofthese compounds were separately extracted using perchloric acid and heattreatments, and then subjected to HPLC analysis after dansylation forfluormetric detection. Free and conjugated forms of putrescine andN-methylputrescine increased in the MPO-suppressed lines R11 and R39. Incontrast, spermidine and spermine were not markedly affected, exceptthat conjugated spermine in a perchloric acid-insoluble fraction waselevated relative to the control. Overall, MPO suppression resulted inincreased accumulation of N-methylputrescine and some polyamines.

EXAMPLE 6 MPO Suppression in Transgenic Tobacco Plants Production ofTransgenic Tobacco Plants

The MPO1 RNAi suppression vector pBI-MPO-Ri was transformed intoAgrobacterium tumefaciens strain EH.4105, which was used to transformleaf segments of tobacco variety K326. Transgenic TO shoots wereregenerated.

To produce transgenic plants, transgenic shoots are transferred torooting medium. Several rooted transgenic plants are transferred tosoil. Transgenic plants are grown at 27° C. under continuous light in agrowth chamber.

Expression of MPO

MPO expression in transgenic plants is analyzed by Northern blotanalysis. The root parts of transgenic and control plants are collectedand immediately stored frozen using liquid nitrogen. RNA is extractedfrom the frozen roots using an RNeasy Midi Kit (Qiagen) according to themanufacturer's protocol, except that polyvinyl pyrrolidone is added to aconcentration of 1% to the extraction buffer. The column operation isperformed twice to increase the purity of the RNA. RNA blotting iscarried out according to the ordinary methods given by Sambrook, J. etal., MOLECULAR CLONING, Cold Spring Harbor Laboratory, Chapter 7 (2001).

The fragment corresponding to the MPO1 nucleotide sequence in the MPO1RNAi suppression cassette in pBI-MPO-Ri is used as the probe template.The template is prepared by amplification from the cDNA clone using PCRprimers. The probe is labeled with ³²P using a Bcabest labeling kit(Takara) according to the manufacturer's instructions. Hybridization areaccomplished using ULTRAhyb (Ambion) as the buffer according to themanufacturer's protocol. Transgenic plants having reduced levels of MPOmRNA compared to the control are selected.

Procedure for Analysis of Alkaloid Levels

The nicotine content in leaves of plants is sampled 36 days aftertransfer to soil. Alkaloids are extracted from the transgenic tobaccoleaves and analyzed, as described above. Lines that show reducednicotine accumulation compared to control (non-transformed lines) areselected.

EXAMPLE 7 Overexpression of MPO1 in Tobacco BY-2 Cells

To examine whether MPO overexpression changes the alkaloid profile, abinary vector containing MPO1 cDNA under the control of the CaMV 35Spromoter was constructed and introduced by Agrobacterium-mediatedtransformation into tobacco BY-2 cells. A full-length MPO1 ORF wasamplified by PCR with primers attached with appropriate restrictionsites with high fidelity KOD-plus DNA polymerase (Toyobo) and subclonedinto multi-cloning sites of pBluescriptII. After sequencing forconfirmation, the ORF was excised and inserted into pBI121 to producepBI-MPO (see FIG. 6B) Agrobacterium tumefaciens strain EHA105 with thevector was used to transform tobacco BY-2 cells and transgenic cellswere selected by kanamycin resistance. Independent transgenic cell lineswere subcultured each week in liquid MS medium supplemented with thesynthetic auxin 2,4-D. Because tobacco BY-2 cells do not produce tobaccoalkaloids under normal culture conditions, four-day-old tobacco cellcultures were rinsed to remove 2,4-D, and were transferred to freshauxin-free MS medium containing 100 μM methyljasmonate, and cultured foran additional 1 day for the RNA and protein analyses, or for 3 days foralkaloid measurements.

Overexpression of MPO1

For RNA gel blotting, total RNA was isolated frommethyljasmonate-treated cells with a RNeasy Kit, separated byelectrophoresis on a 1.2% formaldehyde agarose gel, and transferred ontoa Hybond-N+ nylon membrane (Amersham Pharmatia Biotech). Equal loadingof RNA was confirmed by ethidium bromide staining. The blots werehybridized with a MPO1 cDNA fragment that was labeled with ³²P by usinga Bcabest labeling kit (Takara). Three independent lines (OX4, OX6, andOX14) showed highly increased mRNA accumulation, in contrast to thecontrol cells that showed no detectable MPO mRNA. See FIG. 10A.

The transgenic cell lines OX4, OX6, and OX14 also showed enzymaticactivity of MPO, in contrast to the control cells that showed nodetectable MPO activity. See FIG. 10B.

Alkaloid Profiles

Alkaloid contents in tobacco BY-2 cells after methyljasmonate treatmentwere analyzed. Anatabine was the major alkaloid observed in the controlBY-2 cells, reflecting the very low expression of MPO1. On the contrary,MPO1-overexpressing cell lines accumulated highly elevated levels ofnicotine and reduced levels of anatabine. See FIG. 11. This suggeststhat increased expression of MPO1 facilitated formation ofmethylpyrrolinium cation, resulting a large shift in thenicotine-to-anatabine ratio in favor of nicotine accumulation.

EXAMPLE 8 Overexpression of MPO1 in Tobacco Plants

Description of pBI-QPT-MPO1 Overexpression Vector

The MPO1 cDNA sequence was inserted between the pTobRD2 promoter and thenos terminator to produce an MPO1 expression cassette in whichexpression is controlled by the root-cortex specific promoter. The MPO1expression cassette was cloned within the T-DNA borders of anAgrobacterium binary vector plasmid that contains an nptII selectablemarker cassette within the T-DNA region to produce an MPO1overexpression vector pBI-QPT-MPO1 with the T-DNA region comprising thenptII cassette and the TobRD2-MPO1 expression cassette. See FIG. 6C.pBI-QPT-MPO1 is similar to pBI-MPO1, except that the MOP cDNA isexpressed from the tobacco root cortex specific promoter rather than theCaMV 35S promoter.

Production of Transgenic Tobacco Plants

The MPO1 overexpression vectors pBI-QPT-MPO1 and pBI-MPO1 weretransformed separately into Agrobacterium tumefaciens strain EHA105,which was used to transform leaf segments of tobacco cultivar K326.Transgenic TO shoots were regenerated on selection medium, andtransferred to rooting medium.

Several rooted transgenic plants from independent transgenic lines aretransferred to soil to produce material for further analysis.

Expression of MPO

MPO expression in transgenic plants is analyzed by Northern blotanalysis. The root parts of transgenic and control plants are collectedand immediately stored frozen using liquid nitrogen. RNA is extractedfrom the frozen roots using an RNeasy Midi Kit (Qiagen) according to themanufacturer's protocol, except that polyvinyl pyrrolidone is added to aconcentration of 1% to the extraction buffer. The column operation isperformed twice to increase the purity of the RNA. RNA blotting iscarried out according to the ordinary methods given by Sambrook, J. etal., MOLECULAR CLONING, Cold Spring Harbor Laboratory, Chapter 7(2001)).

The sequence fragment from the start (1 bp) to 413 bp of the MPO1nucleotide sequence (SEQ ID NO: 1) is used as the probe template. Thetemplate is prepared by amplification from the cDNA clone using PCRusing the following primers:

primer 1: 5′-GGGTTCTCATCRCAGCTTTC primer 2: 5′-CCATCTCTGACCTCAGGTGTT

The probe is labeled with 32P using a Bcabest labeling kit (Takara)according to the manufacturer's instructions. Hybridization areaccomplished using ULTRAhyb (Ambion) as the buffer according to themanufacturer's protocol.

Transgenic plants having elevated levels of MPO mRNA compared to thecontrol are selected.

EXAMPLE 9 Overexpression of MPO and PMT and Suppression of QPTDescription of the Vector

A PMT ORF sequence (NCBI accession number D28506) was inserted betweenthe pTobRD2 promoter and the nos terminator to produce a PMT expressioncassette in which PMT is expressed under control of the root cortexspecific promoter. (see WO 2007072224).

A QPT suppression cassette is constructed by cloning two copies of asegment of the QPT cDNA in inverted orientation separated by the Pdkintron operably linked to the TobRD2 promoter and the nos terminatorsuch that the cassette expresses an RNA capable of forming a dsRNAregion comprising a segment of the QPT sequence. U.S. published patentapplications 20060185684.

The PMT expression cassette, the MPO1 overexpression cassette describedin Example 8, and the QPT RNAi suppression cassette are cloned withinthe T-DNA borders of an Agrobacterium binary vector plasmid thatcontains an nptII selectable marker cassette within the T-DNA region toproduce the vector having a T-DNA region diagramed in FIG. 6D.

Production of Transgenic Tobacco Plants

The vector is transformed into Agrobacterium tumefaciens strain EHA 105,which is used to transform leaf segments of tobacco cultivar K326.Transgenic TO shoots are regenerated, and transferred to the rootingmedium. Several rooted transgenic plants are transferred to soil.

Analysis of Alkaloid Levels

Alkaloids are extracted from the leaves of the transgenic tobacco plantsand analyzed, as described above. The alkaloid profile in leaves ofplants sampled 36 days after transfer to soil is analyzed. Transgeniclines that show an increased ratio of nicotine to total alkaloid contentare selected.

TABLE 1 Substrate Specificity of MPO1 and Pig Diamine OxidaseRecombinent tobacco MPO1 Pig kidney diamine oxidase Vmax Km Vmax Km(pkat/mg) (mM) V/K (pkat/mg) (mM) V/K 1,3-Diaminopropane 666 ± 86  0.158± 0.10 4200 N-Methyl-1,3-diaminopropane 1270 ± 131  0.096 ± 0.02 13000r-Butylamine 862 ± 138 0.249 ± 0 18 3500 Putrescine 902 ± 24  0.247 ±0.04 3600 79 ± 7.6 0.228 ± 0.09 350 N-Methylputresine 926 ± 160 0.036 ±0.02 26000  83 ± 13.5 0.657 ± 0.21 120 Cadavarine 715 ± 109 0.362 ± 0.322000 51 ± 9.2 09.15 ± 0.42 55

1. An isolated nucleic acid molecule comprising a nucleotide sequenceselected from the group consisting of: (a) a nucleotide sequence setforth in SEQ ID NO: 1; (b) a nucleotide sequence set forth in SEQ ID NO:3; (c) a nucleotide sequence comprising at least 15 consecutivenucleotides of the nucleotide sequence set forth in SEQ ID NO: 1 or 3;(d) a nucleotide sequence that encodes a polypeptide having the aminoacid sequence set forth in SEQ ID NO: 2 or 4; and (e) a nucleotidesequence that encodes a polypeptide having an amino acid sequence withat least 80% similarity to at least one of SEQ ID NO: 2 and 4 and thathas MPO activity.
 2. A nucleic acid construct, comprising, in the 5′ to3′ direction, a promoter operably linked to a heterologous nucleic acidencoding at least a portion of MPO1 or MPO2 in sense, antisense, orinverted repeat orientation, and a terminator.
 3. A nucleic acidconstruct comprising the nucleic acid molecule of claim 1, wherein saidnucleic acid is operatively linked in sense, antisense, or invertedrepeat orientation to a heterologous promoter.
 4. A plant cellcomprising said nucleic acid construct of claim 2 or
 3. 5. A mutationalvector comprising a sequence of oligonucleotides targeting a regioncomprising 15 consecutive nucleotides of SEQ ID NO: 1 or
 3. 6. A methodof producing a reduced-alkaloid plant, comprising geneticallyengineering MPO suppression in said plant, wherein said engineeringcomprises introducing into a plant cell of said plant the nucleic acidconstruct of claim 2 or
 3. 7. The method of claim 6, wherein the plantis tobacco.
 8. A method for reducing an alkaloid in a tobacco plant,comprising (a) genetically engineering MPO suppression in said plant;and (b) suppressing at least one additional nicotine biosynthesis enzymeselected from the group consisting of aspartate oxidase, quinolinatesynthase, quinolate phosphoribosyl transferase, ornithine decarboxylase,putrescine N-methyltransferase, A622, and NBB1.
 9. A tobacco planthaving genetically engineered suppression of MPO and reduced content ofnicotine.
 10. A tobacco plant comprising a chimeric nucleic acidconstruct, wherein said construct comprises the nucleic acid of claim 1linked to a heterologous nucleic acid.
 11. Progeny of the tobacco plantof claim 9 or 10, wherein said progeny have MPO suppression.
 12. Seedsfrom the tobacco plant of claim
 9. 13. A reduced-alkaloid tobaccoproduct comprising a portion of a tobacco plant of claim
 9. 14. Areduced-alkaloid tobacco product produced from genetically engineeredtobacco having suppressed MPO, wherein said product has a reducedcollective amount of N′-nitrosonornicotine (NNN),4-methylnitrosoamino-I-(3-pyridyl)-1-butanone (NNK), N′-nitrosoanatabine(NAT) and N′-nitrosoanabasine (NAB) compared to a similar tobaccoproduct prepared from a non-genetically engineered control tobaccoplant.
 15. The tobacco product of claim 13, wherein said product is acigarette.
 16. A smoking cessation product comprising a portion of atobacco plant of claim
 9. 17. An MPO1 polypeptide having the amino acidsequence of SEQ ID NO: 2
 18. An MPO2 polypeptide having the amino acidsequence of SEQ ID NO: 4
 19. An isolated MPO enzyme encoded by a nucleicacid sequence selected from the group consisting of: (a) a nucleotidesequence set forth in SEQ ID NO: 1; (b) a nucleotide sequence set forthin SEQ ID NO: 3; (c) nucleic acid sequences which hybridize to at leastone of SEQ ID NO: 1 and SEQ ID NO: 3 under moderate stringency or highstringency conditions and encode an MPO enzyme; and (d) nucleic acidsequences which differ from the nucleic acid sequence of (a), (b), or(c) above due to the degeneracy of the genetic code and encode an MPOenzyme.
 20. A method of producing an MPO enzyme, comprising (a)introducing an isolated nucleic molecule encoding at least one of MPO1and MPO2 into a cell; and (b) growing said cell under conditions suchthat MPO enzyme is produced.
 21. The method of claim 20, wherein saidcell is selected from the group consisting of bacteria, yeast,filamentous fungi, algae, green plants, and mammalian cells.
 22. Amethod for increasing alkaloids in a plant, comprising overexpressingMPO relative to a control plant.
 23. The method of claim 22, furthercomprising overexpressing PMT.
 24. A method for increasing nicotine in aNicotiana plant, comprising overexpressing at least one of MPO1 and MPO2relative to a control plant.
 25. The method of claim 24, furthercomprising overexpressing at least one of QPT, PMT, A622, and NBB1. 26.An increased alkaloid plant produced by any of the methods of claims22-23.
 27. An increased nicotine plant produced by any of the methods ofclaims 22-25.
 28. An increased nicotine product produced from the plantof claim
 27. 29. The product of claim 28, wherein said product isselected from the group consisting of a cigarette, a pharmaceutical, anda nutraceutical.
 30. A method for increasing nicotine and yield in aNicotiana plant, comprising: (a) crossing an increased nicotineNicotiana plant of claim 27 with a high yielding Nicotiana plant; and(b) selecting a progeny plant with increased nicotine and high yield.31. A method for increasing nicotine and yield in a Nicotiana plant,comprising: (a) introducing into a Nicotiana plant a constructcomprising, in the 5′ to 3′ direction, a promoter operably linked to aheterologous nucleic acid encoding an enzyme that increases yield; (b)regenerating transgenic Nicotiana plants from said plant; and (c)selecting a transgenic Nicotiana plant having increased nicotine contentand increased yield relative to a control plant.
 32. An increasednicotine and yield plant produced by the methods of claim
 31. 33. Amethod for increasing the ratio of nicotine to total alkaloids in atobacco plant, comprising: (a) overexpressing at last one of MPO1 andMPO2, (b) overexpressing PMT, and (c) suppressing QPT.
 34. A plantproduced by the method of claim
 33. 35. A product with increasednicotine to total alkaloids produced from the plant of claim
 34. 36. Theincreased nicotine product of claim 28, wherein said product is selectedfrom the group consisting of a cigarette, a pharmaceutical, and anutraceutical.